Cell Survival Protocol *** These are general guidelines for a cell survival, your particular set of experiments may call for adjustments to this protocol. Plating Cells Plate enough cells in 100mm plates so there will be1 million cells on the day the experiment is processed. This needs to be done two or three days before you start the experiment. A typical experiment will be run with 4 drug doses, with 5 radiation doses (all with built-in controls). For example, your 20 plates could be as follows: Control Group with 0, 2, 4, 6, and 8 Gy of radiation; 1µM drug with 0, 2, 4, 6, and 8 Gy of radiation; and a 3 µM and a 10µM Group each with 0, 2, 4, 6, and 8 Gy of radiation. Drug and Radiation Treatments The radiation and drug concentrations will vary, but here is a typical experiment. Day One: Add drug at 10:00 am for a 24 hr drug exposure. Day Two: At 9:30 am irradiate your cells. At 10:00 you should be able to start processing your experiment. Dilute the drug stock in order to add more than 10µl of drug to each dish. Processing Experiment Set-up 1. Make up a plating sheet (see attached example) with the number of cells you want to plate per condition. If you are not sure of the number to plate make a duplicate set of plates for that condition (i.e. E1 and E2). The duplicate set should contain at least 4X the number of cells. 2. Label 1 15ml centrifuge tube for each of your drug/radiation conditions 3. Label 1 counting vial for each condition and fill it with 9.9 ml of Isoton fluid. 4. Label 3 dishes for each condition (label 4 dishes for your control). Label 100mm plates for cell counts of 100,000 to 200,000 cells (do not plate more cells then this). All others can be plated in 60 mm dishes. One plate per condition should have the following information on it: Experiment #, Letter for that condition, treatment dose, cell number you want to plate (i.e. R31, S2, 10C5, 2 x 104 (Numbers and letters are explained on the plating sheet)). The other two plates just need the letter on them. 5. Fill all dishes with media-5ml in the 60 mm dishes and 10 ml in the 100mm. Trypsinizing Cells 1. Check the condition of your cells under the microscope-- it's pointless to start if you don't have any living cells. 2. Aspirate media 3. Add 5 ml PBS 4. Add 5 ml Trypsin (to get cells off dish). Incubate cells for a few minutes; some cell lines take longer than others. 5. Add 5 ml media to all dishes. (sticky cell lines will have to be done one at a time-ie. add media, and immediately pipet into tube of that dish) 6. Using 10 ml bulk pipets, rinse the dishes using a horizontal then circular/vertical motion to help remove cells from dish. Rotate the dish 90 degrees and repeat. Pipet into tubes. 7. Centrifuge 5 minutes, 1000 rpm. Cell Survival Protocol 8. Aspirate supernatant, keep pellet. 9. Add 10 ml media (5 ml if pellet is tiny) and resuspend using bulk pipets. 10. Vortex tubes. Remove 100 µl from tubes to non-sterile counter cups containing 9.9 ml isotonic solution. 11. Count cells twice (Each count measures only 0.5 ml). Making Dilutions & Plating – See plating sheet for examples (The control plate is calculated here) 1. Calculate the number of cells per ml. If the counts were 617 + 646 = 1263 cells / ml in counting vial . This is a 1:100 dilution so there is 126,300 cells/ml in the tube. 2. To make plating easier, we make dilutions of 10,000 cells. (10,000 cells) / (number of cells/ml in tube) = dilution factor (10,000 cells) / (126,300 cells/ml) = 12.63 therefore, I want to add 1 ml from my tube of cells to 11.63 ml of media. 3. If you have 2 x 104 cells or more just do a straight addition of cells from the tube. i. e. Letter D on the plating sheet (20000 cells)/ (51400 cells/ml) = 389 µl of cells/ dish 4. Add the appropriate number of cells to each dish. a) i.e. 250 cells equals 25 µl from dilution. b) Do not add less than 25 µl of cells to a dish. If you need100 cells, make another 1:10 dilution (0.5ml cells from first dilution + 4.5 ml of media). Then you can add 100 µl to each dish. 5. If there are not enough cells to fill at least two plates, split the remaining number of cells into two plates. Make note of the number of cells actually plated. (See J on Plating Sheet) Staining After about 10 days you can stain your plates. There is a staining protocol in the Procedure Book or see below. The colonies need to have at least 50 cells each, but they should still look healthy and stuck to the plate. When colonies are of sufficient size (>50 cells/colony), remove from the incubator. Wear a lab coat and gloves. Label the sides of the dishes with a solvent (alcohol resistant) pen. You can throw away the lids at this point. Add methanol/acetic acid to all the dishes, leaving the medium in the dishes. Point the tip of the squirt bottle at the side, not the bottom of the dish. Add just enough for the medium to turn yellow. Dump all the dishes. Add more Methanol/acetic acid to all the dishes. Use enough to completely cover the bottom of the dish. Let sit 2-5 minutes. Dump the Me/Ac from the dishes and dip the dishes into the beaker of water. Have the water running. Dump and dip 2 dishes at a time instead of dumping all then dipping. Add crystal violet solution to cover the bottom of the dishes. Dump and dip as above. It's important to rinse well at this step. Let the dishes dry open side down. Methanol/Acetic acid Methanol 7 parts, Glacial acetic acid 1 part fume hood! Crystal violet ~1 gram/liter of water It's should be a deep purple. Prepare this in the Cell Survival Protocol Counting Colonies Count the number of colonies per dish with the red counting pen and the dissecting microscope. Write the number of colonies on the bottom of the plate. Only count colonies with at least 50 cells. There needs to be at least 20 colonies per plate to use that condition in the final calculations. The duplicate set of plates do not have to be counted if there are at least 20 colonies in the first set (i.e. E1 and E2). When all the plates are counted, fill the colony number on your plating sheet. Enter the plating sheet information into the computer .