Haynes:FCMamalian FluorGene: Difference between revisions

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(New page: =Detecting Fluorescent Gene Expression= Karmella Haynes 2014<br> REAGENTS: * '''32% Paraformaldehyde''' (EMS Cat # 15714) - 32% solution, EM grade, MeOH-free ** Dilute to final concentr...)
 
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=Detecting Fluorescent Gene Expression=
<- [[Haynes:Protocols | Back to Protocols]]
 
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=Detecting Fluorescent Protein Expression=
Karmella Haynes 2014<br>
Karmella Haynes 2014<br>




REAGENTS:
REAGENTS:
* '''32% Paraformaldehyde''' (EMS Cat # 15714) - 32% solution, EM grade, MeOH-free
* '''FACS Buffer''' - 1% FBS in 1x PBS (stored at 4°C)
** Dilute to final concentration of 1% in 1X PBS just before each use.
 
* '''FACS Buffer''' - PBS with 1% FBS (stored at 4°C)
MATERIALS
* '''10% FB-S''' Saponin Staining/Wash Buffer (Sigma Catalog # S4521) - 10% stock (stored at 4°C)
* '''FACS tubes with strainer caps''' - 6 mL capacity (EMS 64750-25)
** Dilute to final concentration of 0.2% in FACS Buffer just before each use.
* Primary antibody
* Secondary antibody (if primary is not dye-conjugated)




PROTOCOL<br>
PROTOCOL<br>
''This procedure is for two samples. One stained experimental sample and one unstained flow cytometry "blank" control. Scale-up as needed.''
''This procedure is for two samples: one fluorescence-expressing experimental sample and one non-expressing "blank" control. Scale-up reagents as needed for more samples.''
<br>
<br>


'''Harvest the cells''' - do the following in the TC room
'''Harvest the cells''' - do the following in the TC room
# Obtain an ice bucket. Aliquot 5 mL '''FACS buffer''' per sample (10 mL) into a conical tube. Chill on ice.
# Obtain an ice bucket. Aliquot 10 mL '''FACS buffer''' per sample (5 mL per sample) into a conical tube. Chill on ice.
# Harvest ~2x10<sup>6</sup> cells (~1-2 wells of adherent mammalian cells from a 6-well plate) per sample using the standard trypsinization procedure.
# Harvest ~2x10<sup>6</sup> cells (~1-2 wells of adherent mammalian cells from a 6-well plate) per sample using the standard trypsinization procedure.
# Collect the cells in growth medium and transfer to 15 mL conical tubes.
# Collect the cells in growth medium and transfer to 15 mL conical tubes.
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'''Paraformaldehyde Fixation''' - keep the cells on ice as much as possible
'''Flow cytometry''' (single tubes)
# Make fresh '''1% Paraformaldehyde Solution''': add 62.5 μL 32% paraformaldehyde to 1937.5 μL 1x PBS (final volume = 2 mL; 1 mL for each sample)
# Power-on the flow cytometer and allow it to warm-up as needed.
# Label empty 1.5 mL microfuge tubes (one per sample).
# Resuspend each cell sample (disrupted cell pellet) in 500 μL of '''cold FACS Buffer'''. Keep the samples on ice.
# Add 1ml of '''1% Paraformaldehyde Solution''' to the cells in each 15 mL conical, transfer cells to the appropriate labeled 1.5 mL tube, and incubate for '''12 min. on ice'''. (Longer incubations compromise the quality of CV’s in DNA histograms.)
# Label one empty '''FACS tube with strainer cap''' per sample and place them on ice.
# Immediately pellet the cells at room temperature at 1000 rpm for 3 min. Discard the buffer into a conical or beaker (so that you an later discard the paraformaldehyde as has waste). Flick each tube to break up the pellets.
# Using a 1000 μL micropipette, force each 500 μL of cells through a separate, labeled, clean '''FACS tube strainer cap'''.
# Wash the cells 2x with cold FACS buffer: Add 1 mL '''cold FACS buffer''' to the cells. Pellet the cells at room temperature at 1000 rpm for 3 min. Discard the buffer. Flick each tube to break up the pellets. Repeat this process.
# Fill an additional FACS tube with ~2 mL clean water.
<br>
# Take the following (on ice) to the flow cytometer: (1) strained cell samples in labeled FACS tubes, (2) FACS tube with clean water, (3) left-over FACS buffer.
 
# Following the manufacturer's software instructions, enter the appropriate settings per these basic, general guidelines:
'''Saponin Permeabilization'''
## Total cell counts: 5,000 - 10,000 is recommended for mammalian cells. Note: For abundant cell samples, higher counts will be achieved in less time, and will use up very little volume of sample.
# Make fresh '''0.2% FB-S''' for the cells: add 120 μL 10% FB-S to 5880 μL cold FACS buffer (final volume = 6 mL; 3 mL for each sample)
## '''Cell size scatter-plot''': set up a live display window with forward-scatter and side-scatter on the x- and y-axis.
# Resuspend cells in 1 ml '''0.2% FB-S''', added slowly while flicking the sample.
## '''Fluorescence signal histogram''': set up a live display window with the appropriate fluorescence signal on the x-axis (e.g. mCherry/RFP) and cell counts on the y-axis. Set up one per fluorophore (e.g. one for mCherry/RFP and one for GFP if cells express either or both in your experiment)
# Incubate at room temperature for 30 minutes.
## Advanced - '''Dual signal scatter plot''': set up a live display window with one fluorescence signal on the x-axis and the other fluorescence signal on the y-axis.  
# Pellet the cells at room temperature at 1000 rpm for 3 min. Pipette-off the supernatant, leaving a ~100 μL volume of cells plus buffer in the tube. Flick each tube to break up the pellets.
# Following the manufacturer's instructions, load the clean water tube. Run an analysis until no events appear in the gating displays to ensure that the flow stream is clear.
<br>
# Load the "blank" control sample. Run an analysis.
 
# At this point, use the software to create threshold '''gates''':
'''Antibody Staining''' - All staining and washes are to be done in '''0.2% FB-S'''. Do not stain the "blank" control cells. If the Primary antibody is dye-conjugated try to keep the samples protected from light as much as possible.
## '''Cell size scatter-plot''': in this window, draw a polygon/ free-form gate around the cloud of dots ("events" or cells) that clusters near the diagonal slope. Exclude the outliers that fall near the x- or y-axis.
# Make fresh '''0.2% FB-S''' for the antibody dilutions: add 20 μL 10% FB-S to 980 μL cold FACS buffer (final volume = 1 mL)
Analyze on the flow cytometer. Keep the samples on ice until analysis.
# Dilute '''Primary antibody''' in 0.2% FB-S: make 10 μL diluted antibody that is 10X more concentrated than the manufacturer's/ published protocol's recommendation. This is enough for 10 experimental samples.
# Dilute the sample with additional cold FACS buffer if the reads per second exceed the maximum recommended limit.
# Add 1 μL of '''diluted Primary antibody''' <u>to the 100 μL experimental sample ONLY</u>. Incubate for 30 min. on ice.
# Wash the cells 2x with cold 0.2% FB-S buffer: Add 1 mL '''cold 0.2% FB-S buffer''' to the cells. Pellet the cells at room temperature at 1000 rpm for 3 min. Discard the buffer. Flick each tube to break up the pellets. Repeat this process.
# Secondary antibody: If the primary is dye-cojugated, proceed to '''Flow Cytometry'''. If the primary is not dye-conjugated, repeat the steps above for the dye-conjugated secondary. Try to keep the samples protected from light as much as possible.
<br>
 
'''Flow cytometry'''
# Resuspend each cell sample in 500ul of '''cold FACS Buffer'''.
# Using a 1000 μL micropipette, force each 500 μL of cells through a separate, clean FACS tube strainer cap.
# Analyze on Flow cytometer. (keep covered on ice until analysis).
# Dilute the sample with additional cold FACS buffer if the reads per second exceed 250 (Wang lab's bench top flow cytometer).

Revision as of 13:57, 25 June 2014

<- Back to Protocols

Detecting Fluorescent Protein Expression

Karmella Haynes 2014


REAGENTS:

  • FACS Buffer - 1% FBS in 1x PBS (stored at 4°C)

MATERIALS

  • FACS tubes with strainer caps - 6 mL capacity (EMS 64750-25)


PROTOCOL
This procedure is for two samples: one fluorescence-expressing experimental sample and one non-expressing "blank" control. Scale-up reagents as needed for more samples.

Harvest the cells - do the following in the TC room

  1. Obtain an ice bucket. Aliquot 10 mL FACS buffer per sample (5 mL per sample) into a conical tube. Chill on ice.
  2. Harvest ~2x106 cells (~1-2 wells of adherent mammalian cells from a 6-well plate) per sample using the standard trypsinization procedure.
  3. Collect the cells in growth medium and transfer to 15 mL conical tubes.
  4. Pellet the cells at room temperature at 1000 rpm for 3 min.
  5. Aspirate off the growth medium, but leave enough to cover the pellets. Flick each tube to break up the pellets. Chill the cells on ice.
  6. Wash the cells 2x with cold FACS buffer: Add 1 mL cold FACS buffer to each sample. Pellet the cells at room temperature at 1000 rpm for 3 min. Aspirate off the buffer, leaving enough to cover the pellets. Flick each tube to break up the pellets. Repeat this process.


Bring the ice bucket with the cell samples and cold FACS buffer to your bench. The following steps do not need to be carried out under sterile conditions. However, if you do not have any 1xPBS at your bench, please go to the tissue culture room and make an aliquot in a 50 mL conical in the biosafety cabinet, then keep the 50 mL (labeled "non sterile 1xPBS") at your bench. Do not handle dedicated TC reagents outside of the TC room.

Flow cytometry (single tubes)

  1. Power-on the flow cytometer and allow it to warm-up as needed.
  2. Resuspend each cell sample (disrupted cell pellet) in 500 μL of cold FACS Buffer. Keep the samples on ice.
  3. Label one empty FACS tube with strainer cap per sample and place them on ice.
  4. Using a 1000 μL micropipette, force each 500 μL of cells through a separate, labeled, clean FACS tube strainer cap.
  5. Fill an additional FACS tube with ~2 mL clean water.
  6. Take the following (on ice) to the flow cytometer: (1) strained cell samples in labeled FACS tubes, (2) FACS tube with clean water, (3) left-over FACS buffer.
  7. Following the manufacturer's software instructions, enter the appropriate settings per these basic, general guidelines:
    1. Total cell counts: 5,000 - 10,000 is recommended for mammalian cells. Note: For abundant cell samples, higher counts will be achieved in less time, and will use up very little volume of sample.
    2. Cell size scatter-plot: set up a live display window with forward-scatter and side-scatter on the x- and y-axis.
    3. Fluorescence signal histogram: set up a live display window with the appropriate fluorescence signal on the x-axis (e.g. mCherry/RFP) and cell counts on the y-axis. Set up one per fluorophore (e.g. one for mCherry/RFP and one for GFP if cells express either or both in your experiment)
    4. Advanced - Dual signal scatter plot: set up a live display window with one fluorescence signal on the x-axis and the other fluorescence signal on the y-axis.
  8. Following the manufacturer's instructions, load the clean water tube. Run an analysis until no events appear in the gating displays to ensure that the flow stream is clear.
  9. Load the "blank" control sample. Run an analysis.
  10. At this point, use the software to create threshold gates:
    1. Cell size scatter-plot: in this window, draw a polygon/ free-form gate around the cloud of dots ("events" or cells) that clusters near the diagonal slope. Exclude the outliers that fall near the x- or y-axis.

Analyze on the flow cytometer. Keep the samples on ice until analysis.

  1. Dilute the sample with additional cold FACS buffer if the reads per second exceed the maximum recommended limit.
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