RXR α Protocol

Plating Cells:
1. Aspirate media from a 100% confluent small flask of INS-1 832/13 cells
2. Detach cells with 2 mL trypsin.  Let sit in incubator for 5-10 mins (until all cells have detached from flask).
3. Add 8 mL RPMI media.  
4. Mix thoroughly, and take out 0.5 mL from this mixture and put in an Eppendorf tube.
5. Take out 100 μL from this tube and put in a new Eppendorf tube. Dilute with 900 μL 1x PBS (1:10 Dilution)
6. Mix (use vortex) and take out 15 μL per side of the hemocytometer for cell counting.
7.  Add up the counts for both sides and divide by 10 (to get avg. # of cells per square).  Then multiply this by 10 (to account for the 1:10 dilution). Then multiply by 10^4 to get cells per mL (this is telling us the number of cells per mL in the first eppendorf tube).  
8. Next, do (cells/well)/(cells/mL).  Cells per well depends on how many you want.  For this experiment, you want cells/well to be about 1.0x10^5.  This formula will give you mL/well.  So, if my formula gave me 65 uL per well and I was filling 48 wells, I could do (65 uL/well)x60 (which would account for two plates, or 48 wells, plus 12 extra wells just for safety) which would mean I would need 3.6 mL total. I would need to add 435 uL media per well to get to 0.5 mL per well, so I would need (435 uL/well)x60 =26.1 mL total media.  I would take out 26.1 mL media and put it in a boat and take out 3.6 mL from the trypsinized flask and put it in the same boat fill the wells accordingly (0.5 mL per well) with the multi-channel pipette. Make sure to mix the cell mixture frequently using the multi-channel pipette.                                 

Adding Virus – when plating two 24-well plates and
1. Do this at least 12 hours after plating.  Make up a 5 uL virus + 4 mL media mix (if doing two 24 well plates).  Mix the mixture aspirate the media on the first plate.  After mixing, put 0.5 mL in each of the first 4 wells 2.
2. Next, add 2 more mL media to the virus/media mixture and and mix again, and then put 0.5 mL of this mixture into the next four wells.  Do this until you get to the control, where you will simply add 0.5 mL virus-free media per well. Repeat this process for the next plate.
3. Leave virus on for 4 hours, then aspirate all wells and replace with 0.5 mL media per well.
4. Let sit for about another 24 hours, then aspirate all media.  Replace media from apoptosis plate with 0.5 mL 1.0 mM palmitate media per well, and the proliferation plate with 0.5 mL RPMI media per well. Let sit for another 24 hours (need to be pretty exact on this time), and then do cell counts. (In total, 48 hours after putting on virus).