Red Blood Cell Lysis in Digested Tumor Samples for Single-Cell RNA Sequencing

To begin, obtain the filtered and digested tumor sample. Centrifuge the sample at 450 G for five minutes at four degrees Celsius. Aspirate the media without disturbing the pellet and resuspend the pellet in ACK lysis buffer to remove red blood cells and record the volume used.

Repeat the centrifugation and the lysis steps until the sample pellet has no visible red color after centrifugation. Then resuspend the sample pellet in fresh media. Mix 10 microliters each of trypan and blue and the dissociated cells in a 1.5 milliliter tube.

Load 10 microliters on each side of a hemocytometer. to count the cells. Record the cell concentration.

Total live cell count viability percentage, and final media volume after count.