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<h1>Experiment Notes</h1>

  <TR>
     <TH COLSPAN="2"><strong>Table of Contents</strong>
     </TH>
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     <TR>
     <TD>

<A HREF="#scroll1">1. Cellular Uptake Experiment</A> <A HREF="#scroll2">2. Structure Uptake Experiment</A> <A NAME="scroll1"></A><A HREF="#scroll3">3. Cell Viability Experiment</A> <A HREF="#scroll4">4. PTEN Protein Expression</A> <A HREF="#scroll5">5. PTEN mRNA Experiment</A>

</TD>

  </TR>

</TABLE>

<div id="1"> <h3>1. Cellular Uptake Experiment</h3> Treatments:

1. Cells only: Lysotracker stained cells only. 2. Branch (Br scr): Lysotracker stained cells with TOPRO3 stained branch scrambled overhang. 3. Block O (BO scr): Lysotracker stained cells with TOPRO3 stained Block O scrambled overhangs.

Procedure: 1. Staining of structures 1.1 Measure the concentration of structures and concentration of dye needed to stain 50 uL of structures</ul> 1.2 Add correct amount of dye to 50 uL of structure and incubate overnight 2. Staining of cells 2.1 Combine 0.1 uL of 1 mM Lysotracker with 1 mL of Human RPMI media to get final concentration of 100 nM. Incubate at 37 C. 2.2 Count cells. Aliquot 3 samples with ~75,000 cells in each. 2.3 Wash cells with PBS. Resuspend in 200 uL of human media containing Lysotracker dye. Incubate for 1.5 hrs. 3. Structure addition 3.1 After incubation, wash cells with PBS 3.2 Resuspend cells in clear media with 10% FBS. a 175 uL clear media and 25 uL FBS. 3.3 Plate cells in 8 well imaging plate. Add 50 uL of structures to corresponding plates. a. Sample 1 will be unstained cells only (CO Add 50 uL of TAE buffer b. Sample 2 will be stained cells with branch stained with TOPRO. structure concentration: 0.2 nM c. Sample 3 will be stained cells with Block O stained with TOPRO. structure concentration: 0.2 nM 4. TIRF imaging 4.1 Immediately image under TIRF to get t=0 data. After imaging, reincubate for 4 hrs >4.2 Image again to obtain data for t=4 hours</ul>

Results:

t = 0 • Cells only:

<figure> <img src="http://openwetware.org/images/4/48/Exptfig1.png"height="217" width="624"/> <figcaption>Figure 1: Cells stained with Lysotracker only.</figcaption> </figure>

The cells only images at time 0 showed ideal Lysotracker staining, with very little fluorescent signal in the 640 channel. • Branch

<figure> <img src="http://openwetware.org/images/9/9c/Expfig2.png"height="209" width="624"/> <figcaption>Figure 2: Stained cells incubated with stained branch structures.</figcaption> </figure>

</brThe images of cells with branch structures exhibits a strong signal in both the 488 and the 640 channel, confirming the successful staining by both the Lysotracker and the TOPRO3. However, while the Lysotracker signal is present in both cells, the TOPRO3 signal is present only in the cell with a highly granular interior, suggesting a cell nearing death. • Block O

<figure> <img src="http://openwetware.org/images/f/fd/Expfig3.png"height="206" width="624"/> <figcaption>Figure 3: Stained cells with stained Block O structures.</figcaption> </figure>

The cells incubated with Block O showed a weaker Lysotracker staining than the cells with branch. However, the stain was still discernible. As in the branch sample, there was a strong fluorescent signal in the 640 channel which corresponds to stained structures. However, as with the branch sample, the fluorescence originates from dead cell debris rather than any live cells. There was no detectable co-localization of the two signals in any of the samples imaged at t = 0. t = 4 • Cells only

<figure> <img src="http://openwetware.org/images/b/bb/Expfig4.png"height="206" width="620"/> <figcaption>Figure 4: Cells stained with Lysotracker only.</figcaption> </figure>

The cells imaged after the 4 hour incubation displayed a weaker Lysotracker signal than at t = 0. There were weak signals in the 640 channel as well. However, due to the low intensities and high backgrounds, those are most likely residual fluorescence from the dye rather than something extraneous. • Branch

<figure> <img src="http://openwetware.org/images/6/6e/Expfig5.png"height="420" width="624"/> <figcaption>Figure 5: Stained cells incubated with stained branch structures.</figcaption> </figure>

The fluorescent signal from the Lysotracker, while not as strong as at t = 0, was still within ideal intensity. There were strong signals in the 640 channel as well, and the signals originated within viable cells. There were multiple points of co-localization between the 488 and 640 channels. • Block O <figure> <img src="http://openwetware.org/images/9/9a/Expfig6.png"height="423" width="622"/> <figcaption>Figure 6: Stained cells with stained Block O structures.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/7/7e/Expfig7.png"height="424" width="624"/> <figcaption>Figure 7: Stained cells with Block O structures showing structures that were not yet uptaken.</figcaption> </figure>

The images for Block O at t = 4 shared many similar characteristics to those of for the branch, including multiple instances of co-localization between the two channels. However, in certain cases, a strong fluorescent signal was observed in the 640 channel located at the very edge of the cell membrane. This was not observed in any samples containing the branch structure. Discussion: In previous versions of the experiment, high cell concentration has always presented a problem. After attempting the experiment with 250,000 and 100,000 cells, 75,000 cells were used for this experiment. As a result, the cell concentration was ideal and single cells could be analyzed without interference from debris. However, the distribution of cells was not consistent, as there were spots of heavy density and spots where no cells were present. Cell viability was also better than previous experiments, possibly due to the lower cell concentration, and possibly due to an ideal concentration of FBS.

However, in cells where uptake was observed, the cell morphology was less than ideal. Cells displayed a non-circular membrane and granular cytoplasm, signs of an ailing cell. Since structures used did not contain miR-21 complementary overhangs, this could not have been due to the sequestration of miRs. Therefore, either the act of uptake caused the cells to lose ideal morphology, or structures are preferentially uptaken in ailing cells.

Many instances of dye colocalization, and therefore uptake, were observed in both the Block O and Branch treatments. However, more examples of uptake were observed in the Branch treatment. In addition, in the Block O treatment, there were instances where, after 4 hours of incubation, there were structures visible lining the membrane, perhaps in the process of being uptaken. While this is preliminary, the combination of fewer concrete instances of uptake and presence of structures still attempting to be uptaken hints that the Block O structures may not be as preferentially uptaken as the Branch structures, or may be uptaken at a slower rate. Quantitative analysis of uptake must be conducted to draw any conclusions on that matter. For future experiments, overnight time lapse experiments may provide further insight on the mechanism and rate of uptake. The cells could also be nucleofected with fluorescent miR-21 to highlight the mechanism of miR sequestration and combine both structure uptake and cellular uptake in one experiment.

</div>  <div id="4"> <h3>4. PTEN Protein Expression</h3> Treatments: <figure> <img src="http://openwetware.org/images/f/fe/1_treattable.png"height="174" width="434"/> </figure> Procedure: • Cell preparation and incubation: o Count cells and aliquot ~1 million cells per sample. o Wash cells twice and resuspend in 900 uL of cell media. o For cells only samples, add 100 uL of TAE buffer. For other samples, add 100 uL of 1 nM structures to the corresponding sample. o Plate samples in 24 well plate and incubate at 37 C for the desired timepoint (24, 48 or 72 hrs). • Protein extraction o After incubation, wash cells twice in PBS, and resuspend in 100 uL Protein Lysis buffer. Mix vigorously by pipetting. • Lysis buffer preparation: add PMSF in a 1:100 dilution, and protease inhibitor in a 1:200 dilution into lysis buffer. o Incubate cells in Lysis buffer on ice for 10 min. o After incubation, centrifuge sample at 10,000 g for 15 mins. • Protein quantification using BCA assay. o Prepare BCA assay solution for all samples in a falcon tube • 200 uL reagent A for each sample, 1:50 dilution of reagent B. o Prepare standards by serially diluting bovine serum albumin to known concentrations. o In a 96 imaging plate, add 200 uL of BCA assay solution and 10 uL of sample or standard. o Incubate at 37 C for 30 min. o Image plate using a plate reader. • Protein separation using Polyacrylamide gel electrophoresis. o Create a 10% Polyacrylamide gel according to protocol. o Mix 6.5 ug protein from each sample with 10 uL Laemmli solution, and place on heating block at 95 C for 5 mins. o Prepare biotinylated ladder by mixing 1 uL of ladder solution with 15 uL of Laemmli solution. o On gel, load 10 uL kaleidoscope ladder, 11 uL biotinylated ladder, and the entire volume for the samples. Run at 60 V until sample enters gel front. Then, run at 75 V for 2 hours. • Protein transfer to membrane. o Make transfer buffer by adding 100 mL 10x transfer buffer, 200 mL methanol, and 700 mL dH2O. o Cut out membrane of proper size. o Charge membrane by washing in methanol for 15 s, then water for 5 min, then soak in transfer buffer. o Assemble transfer apparatus in correct order. From top to bottom: foam pad, Whatman paper, membrane, gel, Whatman paper, foam pad. • When placing gel, ensure that gel is not dry for too long, and that it lines up with membrane. o Place assembled apparatus into transfer buffer and run in ice at 100 V for 1 hour. • Incubation with primary antibodies (PTEN). o Remove membrane from transfer apparatus. o Wash once with water, and 4-5 times with TBST buffer for 5 min each on a rocker. o Prepare a 1:1000 dilution of PTEN antibody in 5% milk as blocking agent. o After washes, add 10 mL of antibody solution to membrane. o Incubate membranes in antibody solution on rocker at 4 C overnight. • Incubation with secondary antibodies (PTEN). o Remove primary antibody solution from membranes. o Wash 4-5 times with TBST buffer on rocker with 10 min for each wash. o Prepare 1:1000 dilution of secondary anti-rabbit antibodies and 1:2000 dilution of anti-biotin antibodies in 1% milk as blocking agent. o After washes, add 10 mL of secondary antibody solution to membranes. o Incubate on rocker at room temperature for 1 hour. • Imaging (PTEN) o Remove secondary antibody solution from membranes. o Wash 5-10 times with TBST buffer on rocker. o Prepare developing solution. • 4.5 mL ddH2O, 250 uL developing buffer 1, and 250 uL developing buffer 2. o After wash, blot membrane to remove excess liquid and lay membrane on flat surface. o Add developing solution on membrane until uniformly covered. o Leave developing solution on membrane for 20 s, then blot off excess liquid. o Lay membrane on cassette, wrap in plastic wrap, and close cassette to protect from light. o In dark room, place film on top of membrane inside cassette, and let film develop for ~30 s. o Remove film, and place in developer to obtain image.

• GAPDH Detection o Follow same procedure for primary antibody incubation and secondary antibody incubation using the anti-GAPDH antibody instead of the anti-PTEN antibody. For imaging, develop film ~3 s instead ~30 s.

Results:

• Total protein concentrations using BCA protein assay.

<figure> <img src="http://openwetware.org/images/3/31/2bcacurve.png"height="306" width="446"/> <figcaption>Figure 1: BCA standard curve using bovine serum albumin dilutions of known concentrations.</figcaption> </figure> The linear trendline for the standard curve fit well with the experimental data, with a coefficient of correlation over 0.99. However, the resolution of the data did not extend to the lowest concentration tested (0.031 ug/uL). <figure> <figcaption>Table 1: Total protein concentrations in ug/uL in different samples calculated using the BCA protein assay.</figcaption> <img src="http://openwetware.org/images/a/ad/3_table1.png"height="160" width="572"/> </figure> The total protein concentrations for the different samples showed high, random variability.

• Western blot analysis

24 hrs

<figure> <img src="http://openwetware.org/images/4/4e/4fig2.png"height="134" width="438"/> <figcaption>Figure 2: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> The images of the blots were ideal, with specific antibody binding, resulting in clear bands and low background. The 5% milk blocking solution proved to be better than the bovine serum albumin solution, which caused non-specific binding of antibodies, resulting in high levels of uneven background levels. For the PTEN bands, the antibody stained well, with sharp intense bands. The band front was slightly crooked, but was probably the result of loading errors rather than differences in the protein composition. Qualitatively, the Br mir sample displayed the highest concentration of PTEN levels, whereas the cells only sample showed the least. For BO, the scr sample seemed to have higher intensity, suggesting a higher expression of PTEN relative to the mir sample. For the GAPDH bands, the antibody staining was not ideal, since the bands were not sharp and showed variable intensity. The development time was also too high, since the bands bled into each other. In terms of expression, the different samples seemed to show similar levels of band intensity, suggesting that the treatment did not affect GAPDH expression

<figure> <figcaption>Table 2: Densitometric analysis of western blot band intensities to determine PTEN expression relative to housekeeping protein (GAPDH) and different controls.</figcaption> <img src="http://openwetware.org/images/a/a2/5table2.png"height="176" width="458"/> </figure> <figure> <img src="http://openwetware.org/images/c/cb/6fig3.png"height="348" width="496"/> <figcaption>Figure 3: Graph showing relative expressions of PTEN in cells incubated with different structures after 24 hours incubation.</figcaption> </figure>

<figure> <img src="http://openwetware.org/images/0/0d/7fig4.png"height="346" width="494"/> <figcaption>Figure 4: Graph showing relative expressions of PTEN in cells incubated with different structures after 24 hours incubation.</figcaption> </figure> The densitometric analysis confirmed the qualitative assessments made using the blot images. After normalization to the GAPDH band, the highest intensity band was the Br scr, with the lowest intensity being the cells only sample (Table 2). Further normalization to the cells only control showed that all four structure treatments caused a significant increase in the PTEN levels, as shown in Figure 3. However, further normalization to the scrambled overhangs control highlighted the discrepancies between the scrambled and the complementary mir treatments for both structures. For the branch structures, the mir treatment showed an increase in PTEN relative to the scrambled treatment, whereas for Block O the mir treatment showed lower levels of PTEN expression relative to its corresponding scrambled treatment as seen in Figure 4. 48 hrs

<figure> <img src="http://openwetware.org/images/3/33/8fig5.png"height="134" width="438"/> <figcaption>Figure 5: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure>

The characteristics of the blots were at 48 hours were very similar to those at 24 hours, with one significant difference. The sample with the most band intensity was the BO mir sample instead of the Br mir sample. In addition, the BO mir band was visibly more intense than the BO scr sample, while the there was no discernible differences between the intensities of the Br scr and Br mir bands. However, the GAPDH band for the Br scr sample seemed more intense than the that of the Br mir band.

<figure> <figcaption>Table 3: Densitometric analysis of western blot band intensities to determine PTEN expression relative to housekeeping protein (GAPDH) and different controls.</figcaption> <img src="http://openwetware.org/images/d/d3/9table3.png"height="150" width="458"/>

</figure> <figure> <img src="http://openwetware.org/images/0/08/10fig6.png"height="342" width="472"/> <figcaption>Figure 6: Graph showing relative expressions of PTEN in cells incubated with different structures after 48 hours incubation.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/4/48/11fig7.png"height="344" width="494"/> <figcaption>Figure 7: Graph showing relative expressions of PTEN in cells incubated with different structures after 24 hours incubation.</figcaption> </figure> Even though no qualitative conclusion could be drawn about the Branch structures from the blot images, densitometry revealed that the Br mir sample, when normalized to GAPDH, did indeed show a higher PTEN expression relative to the scrambled overhangs expression. Furthermore, in contrast to the 24 hr time point, the BO mir sample also showed a higher level of expression than the BO scr sample. In fact, the increase in expression in the BO sample was greater than that of the Br sample (Figure 7). One continuing trend from the 24 hr time point was the higher level of PTEN expression compared to the cells only sample (Figure 6). 72 hrs:

<figure> <img src="http://openwetware.org/images/9/9e/12fig8.png"height="134" width="438"/> <figcaption>Figure 8: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> At 72 hours, the blot images were significantly different than the other two time points. Once again, the blots were clear with no background. However, in terms of band intensity, the Br scr PTEN band was significantly more intense than all others, where PTEN bands for Br and BO mir samples were non-existent, suggesting that there was no PTEN present in those cells. The GAPDH bands looked similar to those from the 24 and 48 hr time points. Discussion: The BCA protein assay is conducted to measure the concentrations of total protein in unknown protein extract samples so that equal amount of protein can be used for western blot experiments, thus reducing the number of confounding factors. The assay calculates concentration by comparing the absorbance from the unknown sample to a standard curve generated by samples with known concentrations. The resolution of the assay is therefore equal to the range on the standard curve. The standard curve generated for this experiment had an effective range between 0.125 ug/uL and 2 ug/uL, since the 0.031 ug/uL sample did not fit with the linear trend. However, this was not an issue, since all the unknowns measured had concentrations above 0.125 ug/uL.

The branch and Block O structures with miR-21 complementary overhangs were developed with the intention of sequestering free miR-21 from the cell cytoplasm. Since one of most well studied effects of miR-21 over-expression is the suppression of PTEN protein, it was hypothesized that the level of PTEN expression would increase in samples treated with the mir structures compared to controls such as structures with scrambled overhangs or cells incubated without any structures.

At 24 hours, there was a measurable increase in relative PTEN levels in the branch mir treatment compared to the scr or the cells only treatment, suggesting that the branch treatment was successful in suppressing miR-21 and increasing the levels of PTEN protein expression. At 48 hours, both Branch and Block O mir treatments showed an increase in relative PTEN expressions, further reinforcing the positive outlook for the structures.

However, a few issues persisted. At 24 hours, the PTEN expression in the BO scr control was higher than that of the BO mir treatment, suggesting that the mir structure was unsuccessful in sequestering miR-21 at 24 hours, even though they were successful in doing so at 48 hours. While this may seem contradictory, cellular uptake experiments conducted earlier hinted that the Block O structures were possibly uptaken by cells at a slower rate than the branch structures. If so, it is possible that the Block O structures were not present in cells at the threshold levels necessary to cause a significant decrease in miR-21 at 24 hours, but were present at required levels 48 hours after incubation. In addition, even though the BO mir treatment was unsuccessful at 24 hours, at 48 hours it displayed a higher increase in PTEN than the branch structure. This can possibly be explained by the fact that the Block O structure has more overhangs than the branch structure. If, at 48 hours, the concentrations of Block O and Branch had equilibrated to a similar number, the 12 extra complementary overhangs on the Block O structure may explain the greater effect seen in the Block O sample compared to the branch sample.

Another discrepancy was the increased levels of PTEN in the scrambled control relative to the cells only control. Ideally, neither the structures nor the scrambled overhangs on the structures should have any extraneous effect on the cell, so the cells only and the scrambled overhangs controls should effectively be the same. However, since an increase in PTEN was observed, the structures themselves could be having an unexpected additive effect to the regulation of PTEN. To test this theory, further replicates of the present experiment and experiments with structures without any overhangs must be conducted in the future.

Finally, the result for the 72 hour time point study was unexpected, since the PTEN levels in the mir treatment were completely suppressed, whereas the PTEN levels, especially in the Br scr treatment, were highly elevated. Initially, it was though that this could have been caused by a significant loss of viability in the mir treatments due to the increase in PTEN. However, such an event would also have reduced the amount of housekeeping protein (GAPDH) in the sample, which was not observed. Another possibility may be that the apoptotic mechanism itself reduces the amount of PTEN in cells, and thus shows decreased expression levels at extended time points such as 72 hours. However, further replicated of this experiment and further research into literature must be conducted to draw a conclusion about the causes behind such a phenomenon.

This experiment represents only one replicate, and is therefore not statistically significant. To achieve statistical significance, comparable results must be obtained in at least two or more replicates of the same experiment.

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● 18 mM Branch folded 7/16 ● OSU CLL cells ● Qiagen DNeasy Blood and Tissue Kit

Procedure: ● The concentration of Branch was determined using Nanodrop <ul id="list2">○ The concentration was 1.5 nM.</ul> ● 200 uL of OSU CLL cells were washed and incubated in cell media. ● 50 uL of structure was added to the cells, and solution was plated and incubated overnight. <ul id="list2">○ Final volume: 250 uL </ul> <ul id="list2">○ Final concentration of structures: 0.3 nM</ul>

07/30/2014

● The cells were washed once in PBS, then the total DNA was extracted from the cells according to the Total DNA extraction protocol. ● A gel was run with the extracted DNA, purified branch structures, 7249 scaffold, and ladder. Results:

<figure> <img src="http://openwetware.org/images/e/e5/Gel_verification.jpg"height="300" width="300"/> <figcaption>Fig: Gel verification of DNA extraction. From left to right: ladder, scaffold, purified branch structure, DNA extract.</figcaption> </figure>

As the gel shows, the DNA extract had no band that corresponded with the branch purified structure.

Discussion: Since there was no band on the gel corresponding with the folded branch structures, there is no way to confirm whether the structures were uptaken or not and whether the DNA extraction was able to extract the DNA origami structures or not. If structures were uptaken, then the concentration of structures might have been too dilute to register on the gel, since the initial concentration in solution was only 0.3 nM.

For future experiments, the cells should be incubated in a higher concentration of structures. In addition, TEM imaging can be used to determine whether <A NAME="scroll2"></A>there are any structures present in the DNA extracted from the cell.

</div>

<div id="2"> <h3>2. Cellular Uptake Experiment (TIRF) v1</h3> 07/29/2014 Materials: ● 18 mM Branch folded 7/22 ● YoYo1 intercalating dye ● OSU CLL cells ● DiD’ lipophilic membrane stain

Procedure ● The concentration of Branch was determined using the Nanodrop <ul id="list2">○ The concentration was 8.68 nM</ul> ● 50 uL of structure was incubated with 0.31 uL of YoYo1, and stored in the fridge overnight. ● The cells were counted and two samples of ~100,000 cells were prepared. <ul id="list2">○ Sample 1 will be incubated with fluorescent structures.</ul> <ul id="list2">○ Sample 2 will be incubated with non fluorescent structures.</ul> ● The cells were washed, stained with DiD’ and plated in preparation for the uptake experiment. <ul id="list2">○ Final volume of cells was 500 uL.</ul>

07/30/2014 ● The incubated structures were centrifuged, the supernatant removed, and structures resuspended in 1x FOB with 18 mM Mg. ● The fluorescent structures were added to cells in well “F”, and normal structures were added to cells in well “NF” as control. ● The cells were imaged using the TIRF microscope to get a t=0 reading. ● After imaging, the cells were incubated once again. ● After ~4 hours of incubation with structure, the cells were once again imaged with TIRF microscope to get data at t=4 hours. ● After imaging, the cells were incubated again. Results: At t = 0: <figure> <img src="http://openwetware.org/images/0/0e/EN2F1.png"height="203" width="800"/> <figcaption>Fig: TIRF images of cells in well “F” under different wavelengths. From left to right: DIC, Epi 640, TIRF 488, Combined.</figcaption> </figure>

The concentration of cells in both wells were very low.

No viable cells were found in well “NF”, so no images from well “NF” were taken.

In well “F”, a majority of the cells were dead, but there were some cells that were still viable. The cells were attached firmly to the surface, suggesting that the poly-L-lysine worked. The dead cells no longer possessed their spherical shape, and showed compromised cell membranes. There was a lot of background debris, possibly remains of dead cells.

The images at 640 nm were highly saturated, suggesting too high a concentration of DiD’. For live cells, the dye mostly stained the exterior of the cells, and very little of the interior was stained. In dead cells the dye had pervaded throughout. There were significant fluorescence at 488 nm, but only around the dead cells. The signal is hypothesized to have come from autofluorescence by the dead cell debris. The presence of structures could not be confirmed, since no fluorescence at 488 nm was observed outside of the dead cells. At t = 4: <figure> <img src="http://openwetware.org/images/0/01/EN2F2.png"height="272" width="800"/> <figcaption>Fig: TIRF images of cells in well “F” under different wavelengths. From left to right: All, DIC, TIRF 488.</figcaption> </figure>

When imaged under Epi 640, the signal was really bright. This could be because the cells were incubated under DiD for too long, or that the room was too bright.

When imaged under DIC, a few live cells could be observed. Imaging under TIRF 488 showed fluorescence that was localized with the interior of the cell. This could be because the structures were uptaken by the cell, or could be that the plane of view was the top of the cell, and the structures were attached to the cell membrane.

Discussion:

The two biggest problems seemed to be concentration and viability. The low concentration of cells made it more difficult to have a large sample of cell images. This may have been caused by sucking part of the pellet when aspirating, or by miscalculating the initial number of cells, or by incubating the cells in inactivated FBS. To increase cell image sample, the images can be taken at 40x magnification instead of 100x magnification to include more area. More ideally, the initial concentration of cells can be increased from 100,000 cells to 500,000 or 1,000,000 cells.

The low viability of the cells made it more difficult to find viable cells to test for uptake. The fluorescence of dead cells at 488 nm also made it more difficult to isolate structures from cell debris. The low viability may have been because of the number of times the cells were centrifuged, since both samples were centrifuged two extra times and one was centrifuged for three extra minutes. The viability could be increased by handling cells more carefully.

The DiD’ dye did not provide any additional information for our experiment. Since it stains both alive and dead cells, it cannot be used definitively to identify dead cells. The image generated by DiD’ can also be seen by the DIC, so it may be redundant. Instead of a broad staining dye, a viability dye, one that only stains dead cells, may be used instead in the future. To try and confirm whether the structures were actually internalized by the cell, we need to refine our experimental setup. One option would be to take a z-stack image to obtain a 3d model of the cell, and to study whether the fluorescence from the structures originated from the edges of the cell or the center. Another option would be to stain the cells with lysotracker labeling dye, and colocalizing the fluorescence from the dye and the structure. Since lysotracker stains only the lysosome, if the structures are colocalized on the same z-plane, then the <A NAME="scroll3"></A>structures themselves are in the cell.

</div>

<div id="3"> <h3>3. Cellular Uptake Experiment (TIRF) v2</h3> 07/30/2014

Materials:

● 20 mM Branch with poly-T overhangs ● OSU CLL cells ● YoYo1 intercalating dye ● TO-PRO-3 intercalating dye ● Lysotracker Green labeling dye

Procedure: ● Staining of structures: <ul id="list2">○ Measured the concentration of structures <ul id="list2">■ concentration: 10.83 nM</ul></ul> <ul id="list2">○ Calculated the amount of dye required to stain 50 uL of structures <ul id="list2">■ YoYo1: 4 uL</ul> <ul id="list2">■ TO-PRO-3: 0.39 uL</ul></ul> <ul id="list2">○ Combined 50 uL of structure and appropriate amount of dye and</ul><ul id="list2"> incubated in fridge overnight.</ul> ● Staining of cells: <ul id="list2">○ Combined 7.5 uL of Lysotracker dye with 1.5 mL of Human RPMI to </ul><ul id="list2"> get final dye concentration of 50 nM. Warmed media to 37 C.</ul> <ul id="list2">○ Counted cells and aliquoted ~500,000 cells into 3 tubes: control,</ul><ul id="list2"> YoYo, and ToPro.</ul> <ul id="list2">○ Washed cells twice with PBS.</ul> <ul id="list2">○ Resuspended cells in warm media containing Lysotracker dye.</ul> <ul id="list2">○ Plated cells in an 8 well plate and left to incubate overnight. <ul id="list2">■ Plated in 3 different wells: “Y” for YoYo1 stained cells, “T” for</ul><ul id="list2"> TOPRO3 stained cells, and “Ctrl” for unstained cells.</ul></ul> 07/31/2014 ● Removed cells from plate, washed them twice with clear media without FBS. ● Replated cells in aforementioned clear media. ● In well “Y”, added 50 uL of structures stained with YoYo1. <ul id="list2">○ Final concentration of structures: 0.656 nM.</ul> ● In well “T”, added 50 uL of structures stained with TOPRO3. <ul id="list2">○ Final concentration of structures: 0.656 nM.</ul ● In well “Ctrl”, added 50 uL of unstained structures. <ul id="list2">○ Final concentration of structures: 0.656 nM.</ul> ● Immediately imaged cells with TIRF microscope to obtain data for time=0. ● After imaging, incubated cells for ~7 hours. ● After incubation, imaged cells again with TIRF microscope to obtain data for time = 7 hours.

Results

t = 0

● “Ctrl” Well:

<figure> <img src="http://openwetware.org/images/4/4e/3f1.png"height="463" width="680"/> <figcaption>Fig: TIRF image of cells stained with Lysotracker Green after initial incubation with unstained structures. From left to right: composite image, 640 nm channel, 488 nm channel (top row); DIC, 561 nm channel (bottom row).</figcaption> </figure>

The cell concentration in the plate was slightly higher than ideal. A majority of the cells in the plate were viable. However, due to the high concentration of the cells, it was difficult to find a frame with only live cells in it.

The viable cells were perceptibly stained with the Lysotracker dye. However, the staining was irregular and the signal was weak. There was also bleedover into the 560 channel, and the signal in the 560 seemed stronger than that in the 488 channel. The images were also not perfectly in focus, since some of the fluorescence seemed diffuse.

The fluorescence in the 488 channel was localized within the cells, and there was no fluorescence in the 640 channel.

● Well “Y”: <figure>

</br><img src="http://openwetware.org/images/d/dc/3f2.png"height="463" width="680"/>

<figcaption> Fig: TIRF image of cells stained with Lysotracker Green after initial incubation with structures stained with YoYo1. From left to right: composite image, 640 nm channel, 488 nm channel (top row); DIC, 561 nm channel (bottom row).

</font></figcaption>

</figure>

The cell concentration was similar to that in plate “Ctrl”. It was also difficult to find frames with only living cells.

The staining of the cells by Lysotracker was much more apparent at 561 nm, even though ideal absorbance of Lysotracker is around 488 nm. The 488 nm signal seemed very diffuse, with the highest intensities colocalized with areas outside the cells, or within dead cells. The 640 channel showed no fluorescence.

● Well “T”: <figure>

</br><img src="http://openwetware.org/images/c/ce/3f3.png"height="463" width="680"/>

<figcaption> Fig: TIRF image of cells stained with Lysotracker Green after initial incubation with structures stained with TOPRO3. From left to right: composite image, 640 nm channel, 488 nm channel (top row); DIC, 561 nm channel (bottom row).</figcaption> </figure>

The condition and concentration of the cells were similar to that of the other 2 wells.

The staining of the lysotracker was significantly noticeable at 488 nm, and there was no bleed over to 561 nm. The fluorescence at 488 nm was colocalized within the cells only, and there were spikes in the intensity corresponding to possible lysosomes within the cell.

Even though there was no bleed over from the Lysotracker, there were still high signal intensities in the 561 channel. These signals were colocalized with dead cells instead of living ones. The 640 channel also showed slight fluorescence, once again corresponding to the dead cells.

t = 7 hours

● “Ctrl” Well:

</br><img src="http://openwetware.org/images/9/96/3f4.png"height="463" width="680"/>

<figcaption> Fig: TIRF image of cells stained with Lysotracker Green 7 hours after initial incubation with unstained structures. From left to right: composite image, 640 nm channel, DICl (top row); 561 nm channel, 488 nm channel (bottom row).</figcaption> </figure>

After 7 hours, the condition of the cells were much worse. The concentration of cells was still high, but most were in some state of necrosis. As such, it was very difficult to find viable cells to image.

The live cells were still mostly stained with Lysotracker green. The dead or dying cells seemed to have a much weaker signal. The 488 channel showed clear, focused images of staining that colocalized with the interior of the live cells. However, there was also fluorescence in the 561 and 640 channel, often roughly colocalized within live cells, but also present in dead ones and in solution. The signal was weaker and more diffuse than that of the 488 channel.

● Well “Y”:

</br><figure>
</br><img src="http://openwetware.org/images/c/cf/3f5.png"height="463" width="680"/>

<figcaption>Fig: TIRF image of cells stained with Lysotracker Green 7 hours after initial incubation with structures stained with YoYo1. From left to right: composite image, 640 nm channel, DICl (top row); 561 nm channel, 488 nm channel (bottom row). </figcaption> </figure>

The staining by Lysotracker green was very weak and diffuse. Extremely high intensity signals colocalized with dead cells saturated all other possible signals in the 488 nm channel, making it difficult to locate any other fluorescence.

The 561 and 640 channels also produced fluorescence that correlated to the same cells. Both signals were diffuse, but the signal in the 640 channel was weaker than the 561 channel. The signals were not colocalized with the signal from the 488 channel.

● Well “T”:

</br><figure>
</br><img src="http://openwetware.org/images/2/2c/3f6.png"height="463" width="680"/>

<figcaption>Fig: TIRF image of cells stained with Lysotracker Green 7 hours after initial incubation with structures stained with TOPRO3. From left to right: composite image, 640 nm channel, DICl (top row); 561 nm channel, 488 nm channel (bottom row). </figcaption> </figure>

There were weak but sharp signals in the 488 nm channel from the Lysotracker dye. The signals were located within viable cells, though all the viable cells in the frame did not express a signal. The signal was significantly weaker than during the initial imaging.

There was fluorescence in both the 561 and 640 channel. The signals were colocalized with each other. The 640 nm channel presented high intensity signals from within dead cells, which made imaging fluorescence within live cells more difficult. There was no colocalization between signals, in the 488 and 640 channel. However one cell in the frame showed fluorescence from both the 488 and 640 channels.

Discussion:

The concentration of cells were much higher than that observed in experiment v1, with much fewer dead cells. This is most likely a result of using 500,000 cells instead of 100,000, but could also the result of preserving the pellets during washes, and incubating the cells with activated FBS. However, the concentration was slightly higher than ideal, since it was difficult to image small groups of cells, or image frames with no dead or dying cell present. Since overnight incubation in cell media should promote cell division, a smaller starting concentration should be used for future experiments.

After 7 hours, the number of viable cells dramatically decreased in all 3 plates. It was determined that this was most likely due to the additional incubation in clear media without any FBS. This problem could be corrected by incubating the cells in FBS media in between the images. However, since images are best taken in clear media, the cells would have to be spun down and resuspended in a different media after each data collection phase, thus removing the structures from the solution. Another option would be to use different wells for different data collection times. For example, well A could be used for initial measurements, and well B could be used for measurements after 7 hours. This would remove the problem of having to resuspend the same cells multiple number of times. However, since they are different samples, the data may not be as accurate. Finally, the cells could be incubated in clear media with FBS. Since FBS is tinted, the media would no longer be clear, but the slight tint may not interfere with imaging.

The Lysotracker staining during both the initial and final imaging was weaker than expected. This could be caused by a low initial dye concentration, or due to long incubation times. For future experiments, the dye concentration should be increased to ~60 nM. In addition, while the protocol calls for 20 minutes to 2 hours of incubation, our cells incubated in the dye for ~12 hours. This was because the cells were stained the night before the experiment, and the protocol warned of even further weakening of signals and cell blebbing if the cells were incubated for extended periods without the dye. For future experiments, the cells should be dyed at most 2 hours before imaging.

For the unstained structures, we hypothesized that there would be no fluorescence in the 561 for 640 channels, since the only fluorescent substance in the sample was the Lysotracker dye. If there was fluorescence at 561 nm, it would be colocalized with the 488 fluorescence, since the signal would come from the Lysotracker dye bleeding over. During initial imaging, we did not observe any fluorescence at 640 nm. There was fluorescence in the 561 channel, and it was weaker than, anc colocalized with, the 488 nm channel. However, in the t=7 images, there was fluorescence in both 561 and 640 channels, and neither was well colocalized with the 488 channel. The reason for fluorescence, especially at 640 nm, could not be determined, but it could have been caused by impurities in our solution, or auto-fluorescence of dead cell debris. For future experiments, a sample of unstained cells and structures should be prepared to isolate which signals could be caused by autofluorescence.

Since both YoYo1 and Lysotracker have similar excitation wavelengths, we hypothesized that using them concurrently would not yield good results. This was supported by our experiment, which showed very diffuse fluorescence at 488 nm. In addition, the emission intensity of YoYo1 seemed to be much higher than that of the Lysotracker. This made it very difficult to pinpoint on fluorescence by the Lysotracker, since the signal from the YoYo1 saturated the total signal. Therefore, for future experiments, YoYo1 should not be used in conjunction to the Lysotracker dye. Since TOPRO3 and Lysotracker Green have excitation wavelengths at opposite ends of the spectrum, we anticipated better results with the TOPRO dye. Initial images showed clear staining by the Lysotracker dye, but the signal at 640 nm was very weak. However, it was later realized that the weak signal was caused due to a slight malfunction in the microscope rather than the sample. In fact, the fluorescence readings from t=7 displayed high intensities, showing that the structures were successfully intercalated with the TOPRO dye. However, there were two problems associated with the dye. The first was that the signals at 561 nm seemed to correspond directly with the 640 signal. Since it is very rare for a fluorophore to bleed down the spectrum, the cause for the similar fluorescence observed at 561 nm could not be determined. The second problem was the high intensities in dead cells during the t=7 data collection. The DNA structures are easily integrated into dead cell matter, and the significant decrease in cell viability after the 7 hour incubation increased the amount of dead cells in solution. When imaged at 640 nm, the signals from these dead cells would be so high that it would saturate the rest of the signal, making it difficult to isolate any other signal. This phenomenon of high structure affinity to dead cell matter could pose the additional problem of pulling structures out of the solution, leaving less to be uptaken by living cells. For future experiments, measures should be taken to maintain the viability of cells so that the structures are not disproportionately absorbed into dead cells.

The most definitive proof of cellular uptake from this experiment would be colocalization of signals from the Lysotracker dye and the intercalating dye, since that would suggest that the two dyes are occupying the same lysosome. While there were examples of both signals within a single cell, perfect colocalization was not observed in any image. Therefore, while there is a high probability, we cannot definitively conclude that our structures were indeed uptaken by the cells. In the future, the image could be better focused to help identify individual signals, which would make determining colocalization easier. Optimizing the experimental conditions, such as dye concentration and cell incubation conditions, will also increase the probability of observing colocalization.

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@@ Line 306: / Line 306: @@
 <figcaption><font size="2">Figure 7: Graph showing relative expressions of PTEN in cells incubated with different structures after 24 hours incubation.</font></figcaption>
 </figure>
-</br></br>Even though no qualitative conclusion could be drawn about the Branch structures from the blot images, densitometry revealed that the Br mir sample, when normalized to GAPDH, did indeed show a higher PTEN expression relative to the scrambled overhangs expression.  Furthermore, in contrast to the 24 hr time point, the BO mir sample also showed a higher level of expression than the BO scr sample. In fact, the increase in expression in the BO sample was greater than that of the Br sample (Figure 7). One continuing trend from the 24 hr time point was the higher level of PTEN expression compared to the cells only sample (Figure 6).
+</br>Even though no qualitative conclusion could be drawn about the Branch structures from the blot images, densitometry revealed that the Br mir sample, when normalized to GAPDH, did indeed show a higher PTEN expression relative to the scrambled overhangs expression.  Furthermore, in contrast to the 24 hr time point, the BO mir sample also showed a higher level of expression than the BO scr sample. In fact, the increase in expression in the BO sample was greater than that of the Br sample (Figure 7). One continuing trend from the 24 hr time point was the higher level of PTEN expression compared to the cells only sample (Figure 6).
 </br></br><u>72 hrs:</u>
@@ Line 315: / Line 315: @@
 <figcaption><font size="2">Figure 8: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</font></figcaption>
 </figure>
-</br></br>At 72 hours, the blot images were significantly different than the other two time points. Once again, the blots were clear with no background. However, in terms of band intensity, the Br scr PTEN band was significantly more intense than all others, where PTEN bands for Br and BO mir samples were non-existent, suggesting that there was no PTEN present in those cells. The GAPDH bands looked similar to those from the 24 and 48 hr time points.
+</br>At 72 hours, the blot images were significantly different than the other two time points. Once again, the blots were clear with no background. However, in terms of band intensity, the Br scr PTEN band was significantly more intense than all others, where PTEN bands for Br and BO mir samples were non-existent, suggesting that there was no PTEN present in those cells. The GAPDH bands looked similar to those from the 24 and 48 hr time points.
 </br></br><strong>Discussion:</strong>
 </br>The BCA protein assay is conducted to measure the concentrations of total protein in unknown protein extract samples so that equal amount of protein can be used for western blot experiments, thus reducing the number of confounding factors. The assay calculates concentration by comparing the absorbance from the unknown sample to a standard curve generated by samples with known concentrations. The resolution of the assay is therefore equal to the range on the standard curve. The standard curve generated for this experiment had an effective range between 0.125 ug/uL and 2 ug/uL, since the 0.031 ug/uL sample did not fit with the linear trend. However, this was not an issue, since all the unknowns measured had concentrations above 0.125 ug/uL.

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