TIL isolation by Ficoll‐Paque density gradient centrifugation

TILs from 4T1 and MC38 tumors were isolated as described here (Tan & Lei, 2019). Briefly, after indicated antibody treatments mice were euthanized. Next, tumors were harvested using sterile scissors and forceps. After excision, tumors were minced into small pieces in RPMI‐1640 media using two single‐edged razor blades. Small tumor pieces were transferred to a 70 μm cell strainer in RPMI‐1640 media. A rubber plunger of a syringe was used to mesh the dissociated cells through the cell strainer and cloudy media (that contained dissociated cells) was collected onto a sterile labeled 50‐ml conical tube. Tubes were filled with 30 ml of RPMI‐1640 media at room temperature (18–20°C). Immediately before the addition of Ficoll‐Paque media, single‐cell suspension was well mixed with 25‐ml pipette. Thoroughly mixed 10 ml of Ficoll‐Paque media was carefully poured in the bottom of the tube to form a layer of Ficoll‐Paque below the cell suspensions without mixing the cell suspension. Tubes were centrifuged at 1,025 g for 20 min at 20°C with slow acceleration and without applying any brake. Twenty millilitre of the upper layer of media was discarded to a waste bottle from the tube. Layer of mononuclear cells that contained TIL was transferred to a sterile labeled 50‐ml conical tube using a sterile pipette, along with the remainder of the media above the Ficoll‐Paque. TILs were washed three times using 40 ml of complete RPMI media each time. After final wash with RPMI media, isolated tumor infiltrated leukocytes (TILs) were subjected for flow cytometry (FACS) analysis directly or with anti‐CD3 antibody (OKT3) incubation with fluorescently labeled CD4, CD8 and CD45, IFN‐γ etc antibodies. antibodies as described in the text and here (Whitford et al, 1990).