Difference between revisions of "20.109(S14):DNA repair assays(Day6)"

Introduction

Protocols

Part 1: Prepare cells for flow cytometry

1. Begin by briefly looking at your cells under the microscope. Do the cells in any wells appear less dense or less healthy than in others? Note down any such observations.
2. Aspirate the media from each well according to the protocol below.
   • As you work, tip the plate down a little to pool the media at the bottom of each well.
   • Place your aspirator at the bottom of a well, and suck up the media. Remove all the liquid, but remove the aspirator promptly after that or you may damage/aspirate some cells.
   • Before moving to the next (non-duplicate) well, dip your aspirator briefly (less than a second!) in ethanol. Then hold the Pasteur pipet up to dry and count to 3.
   • Alternatively, you may use a fresh yellow tip on the end of the Pasteur pipet when changing samples.
3. Gently distribute 0.5 mL of warm PBS to each well, using a 2 mL serological pipet.
   • In other words, don't blast the liquid right at your cells.
4. Repeat the aspiration step, again using the ethanol to clear the pipet between samples.
5. Add 0.25 mL of phenol-red-free trypsin/EDTA to each well, using a P1000.
6. Incubate for about 3 min, until cells are rounding up and starting to come off.
7. Meanwhile, label your pre-chilled flow cytometry tubes according to the tables below.
8. Distribute 0.25 mL of media to each well. You are welcome to use the same pipet tip for each well, as long as you don't touch the tip down into the trypsin/well!
9. One well at a time, follow the protocol below to transfer a uniform cell suspension to the appropriately labeled flow cytometry tube.
   • Pipet the full 0.5 mL of solution up and down about four times. Make sure to vary where your tip is in the well, concentrating on the four "edges" of the circle (if the circle were, you know, a square) to more fully resuspend the sticky cells. Check the first well or two under the microscope to be confident of your technique. You should see very few cells.
   • On the final resuspension, have your partner hand you the filter-topped flow cytometry tube. Press the blue tip gently against the top of the filter, and expel the cells. You should see the liquid go right through; if you feel resistance, ask for help.
   • Each tube should be immediately returned to your ice bucket.

Sample order for each team: K1 intact, K1 intact duplicate, C401 intact, C401 intact duplicate, xrs6 intact, xrs6 intact duplicate, K1 damaged, K1 damaged duplicate, C401 damaged, C401 damaged duplicate, xrs6 damaged, xrs6 damaged duplicate.

Part 2: Plate irradiated cells for inhibitor dose response

For next time

Reagent list

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