Demonstrated Protocol, Last Modified on March 31, 2021, Permalink
This Demonstrated Protocol outlines best practices for enriching the percentage of Cluster of Differentiation 3 positive (CD3+) T cells obtained from dissociated tumors in preparation for use in 10x Genomics® Single Cell Protocols.
While this Protocol is demonstrated with tissue matched dissociated primary tumor cells and peripheral blood mononuclear cells (PBMCs) from a clear cell renal carcinoma (CCRC) patient, it has also been demonstrated with dissociated primary tumor cells from colorectal cancer (CRC) and renal cell carcinoma (RCC) patients, and with lymph node cells from melanoma patients. This Protocol may also be used as a basis for enrichment of the CD3+ T cell population from other dissociated tissues as well as other primary cells in preparation for use in 10x Genomics® Single Cell Protocols. Modifications to this Protocol may be necessary for other sample types (e.g. media type, resuspension buffer, centrifugation speed and time).
This demonstrated protocol is optimized for counting cells in the range of 700-1200 cells/µl. If using the Single Cell 3' LT v3.1 (low throughput) application, ensure cells are counted as indicated in this protocol and then diluted to the LT specific optimal loading concentration of 100-600 cells/µl using the Cell Dilution Overview in the LT User Guide.