My aim is to isolate TILs from B16F10 tumor. I used Discontinuous method using 80% and 40 % percoll.
I get a concentrated tumor cells above 40% percoll as shown in picture.
The tumor cells are very sticky, and stick to tips when I try to remove them.
Has any one tried isolating TIL from B16F10 tumor?
I have isolated TILs from other syngeneic tumours but I know that B16F10 tumours can be a tricky tumour to work with. I have used the miltenyi gentleMACS kit and that worked well but I know it can be very expensive. You could try coating your tips with EDTA before removing the cells, then washing with PBS and FCS / EDTA? Hope this helps, good luck.
Hi Jian Li, I tried lot of variations using percoll but nothing worked well with B16F10 tumors due to sticky nature of the cells. I finally used miltenyi gentleMACS kit to isolate TILS.
Hi Manoj, I have tried to isolate TILs from B16OVA I hope it help. First, I used 40% percoll and centrifuge at 300g RT for 10 minutes (accel/brake off), after centrifugation aspirate the upper layer and resuspend the pellet with 5 ml of 40% percoll, after that underlay with 3 ml of histoplaqye1083 and centrifuge at 800g RT for 15 minutes (accel/brake off). Finally, aspirate 2 -3 ml of upper layer (Debris and dead cell) and collect 2-3 ml of interface (TILs).
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