Talk:20.109(S14):Preparing cells for analysis (Day4)

Protocols

If you got to go to the TC room first on Day 2, you will go in the second cohort today (and vice-versa). If you are in the second group, you may use the time that you are waiting to work on your FNT or the optional data analysis, but also be sure to prepare your RNase-free area, label tubes that you will need, etc., before heading into TC.

Part 1: Prepare cell lysates

You will prepare cell-bead samples in three different ways: one will allow you to count your cells, and is suitable for RNA preparation, while the other two will involve more stringent bead/matrix dissolution for better protein or proteoglycan recovery. Split up the work with your partner whatever way is most convenient. Remember to label your samples carefully at every step.

If your beads are small and/or fragile, you may need to skip some rinsing steps today. Please also ask the teaching faculty for advice on working with your beads.

1. Before proceeding, briefly observe the cell-bead constructs under the microscope and note any changes from Day 3.
   • Let the teaching faculty know if you have difficulty focusing within a bead.
2. Remove the culture medium from each of your samples. Be careful not to suck up the beads; it will help to use a serological pipet just as you did when washing your freshly synthesized beads. Tipping the plate will help the beads settle in a cluster and allow you to remove medium elsewhere.
   • A 5 mL pipet size should work well for rigid beads, while for more delicate beads, you should use a 2 mL serological pipet or even a P1000. If your beads are falling apart, you can transfer the beads according to steps 3-5 below without trying to remove medium first.
   • If you are concerned about your bead amount, talk to the teaching faculty. You might skip the proteoglycan assay and focus on the other two instead.

     

     Bead preparation for three assays.

3. About 1/3 of your beads will be used to measure protein content: move these to an eppendorf tube. The goal is about 10-15 (2-3 mm) beads per tube.
   • For large beads (4-5 mm), you might use only 5-10 beads, and for very small beads (<1 mm), you might use 20 or more.
4. Another 1/3 will be used to measure proteoglycan content; these whole beads can also be moved to an eppendorf tube.
5. The final 1/3 will be used to isolate RNA. Using a sterile spatula, transfer the beads into a fresh well of your 6-well plate. This transfer step is to exclude any cells that are growing on the bottom of the plate (as opposed to actually in the beads) from analysis.

Samples for RNA Isolation

1. Rinse the transferred bead-cell constructs with 4 mL of warm PBS, then aspirate the buffer.
   • If your beads are very fragile, you might want to skip the PBS rinse, and directly proceed to step 2.
2. Add 3 mL of pre-warmed EDTA-citrate buffer, and incubate at 37 °C for 10 min.
   • Meanwhile, prepare the beads for the protein and proteoglycan assays as described below. All the materials that you need are in eppendorf tubes in the fridge.
3. Now recover your cells:
   • Add 3 mL of warm complete culture medium, pipet up and down to break up the beads (you may find this easier with a 1 mL pipetman rather than a serological pipet), and transfer to a 15 mL conical tube.
   • Spin the cells down at 1900g for 6 min using the centrifuge that is in the TC room.
4. Resuspend in ~ 1-1.5 mL of culture medium, and write down what you use. Mix thoroughly by pipetting, then set aside a 20 μL aliquot of your cells for counting, and put the rest of the cells into another eppendorf tube.
   • If you have very few cells based on your Day 3 observations and/or having very few beads, you might consider skipping the cell count, and instead keeping all of the cells for RNA isolation. If you have too few cells to get a reliable cell count, you are not losing valuable information for your report in any case. And if you have so few cells that taking some of them for a count compromises your other data, then that outcome would not be preferable to missing the cell count.
5. While one of you begins the spin in the main lab (see Part 2), the other should count your cell aliquot similarly to how you did on Day 2. This time, mix 10 μL of Trypan blue with your 20 μL of cells rather than using 90 μL, so we can conserve cells. Separately calculate the approximate numbers of live (yellowish) and of dead (blue) cells.
   • Recall that you must multiply by 10,000 (and your dilution factor) to convert a hemacytometer cell count to a live cells/mL concentration.

Samples for Protein Extraction

1. Per eppendorf tube (typically 10-15 beads), add 133 μL of cold EDTA-citrate buffer (not from the warm conical!), and pipet up and down for 20-30 seconds to dissolve the beads. Be thorough while limiting bubbles as best you can. The resulting solution may be viscous.
2. Pipet 33 μL of 0.25 M acetic acid into each eppendorf tube.
3. Finally, pipet 33 μL of 1 mg/mL pepsin (in 50 mM acetic acid) into each tube and mix well.
4. Move your eppendorf tubes into the rack in the 4 °C fridge. Tomorrow the teaching faculty will move them to an elastase solution (also at 4 °C) to break down the polymeric collagen to more readily measured monomeric collagen.

Samples for Proteoglycan Extraction

1. Soak your beads for a few minutes in pre-warmed PBS, and then remove as much of the PBS as possible. Shoot to have as little pink tint to the beads as possible, as it is known to interfere with the proteoglycan assay. Repeat the soak step a second time with fresh PBS, if it seems to help.
2. Add 250 μL of papain solution to your beads. The papain is in an EDTA-citrate buffer base.
3. When the first partner goes to the main lab, s/he should take this tube to the 60 °C heat block. After 24 hours, the samples will be moved to the freezer.