Sean Lauber:Preparing LLC or B16 cells for injection

NOTE that if you are using LLC-Fluc cells, you MUST culture the cells with 3 ug/ml of puromycin (in the same box with fungizone). For every 10 ml of media, add 6 ul of the puromycin solution (stock is 5000 ug/ml). You do not need to supplement the freezing media with this.

For B16 cells, each mouse is injected with 1 x 10^5 cells For LLC cells, each mouse is injected with 1 x 10^6 cells

A single confluent T150 gives about 5 million cells for both B16 and LLC cells.

1) Thaw the LLC or B16 cells and culture in 10% FBS/DMEM until confluent (about 48 hours) in a T75

2) Split the cells 1:2 into a T150 and incubate for another 24 hours

3) The media from the cells was discarded

4) The cells were washed with 5 ml of PBS

5) 1 ml of trypsin was added and the flasks were moved periodically to keep the surface wet

6) After 3 (LLC) or 4 (B16) min at RT, the flask was knocked firmly 4 times on its side to dislodge the cells, then after another minute was knocked 4 times again before being stopped with 9 ml of 10% FBS DMEM. The solution was pipetted off the surface of the flask twice fully (10 ml pipette)

7) The 10 ml of cell solution was collected in a 50 ml falcon tube and the plate was washed with a further 5 ml of PBS and collected

8) The cells were spun down at 1000 RPM as before, the supernatant discarded, and the cells were resuspended (by tapping) in the residual volume

9) To the side wall of the tube, 50 ml of RT PBS was added to the tube, then was resuspended twice fully with a 25 ml pipette

10) The cells were spun down as before, and washed with 50 ml PBS for a second time

11) After spinning down the cells, the cells were resuspended in 2 ml PBS (5 full resuspensions with a 5 ml pipette, SLOWLY) and kept on ice

12) Two separate 10 ul aliquots were taken and used to count the cells and determine viability using 10 ul trypan blue and 30 ul PBS

13) Dilute the cells appropriately (use the template I have on the main page for help)

14) Inject the cells by tail vein (in 0.4 ml - or whatever volume the injector wants to use)

15) One day after injection, intubate the mice with adenovirus expressing YFC (your favorite cytokine)