Methanol Fixation of Cells for Single Cell RNA Sequencing

Demonstrated Protocol, Last Modified on August 9, 2021, Permalink


20200312_CG000136 Methanol_Fixation_RevDtoRevE.pdf

If you are interested in methanol fixation as a means for processing infectious samples, you may be interested in our page on Processing Samples Containing Infectious Agents using the 10x Genomics Chromium Controller and Protocols.

This protocol outlines methanol fixation and rehydration of single cell suspensions for use with 10x Genomics Single Cell protocols. The protocol was demonstrated with Jurkat T lymphocytes, embryonic brain cells, and human peripheral blood mononuclear cells (PMBCs). Additional optimization may be required when working with other cell types (e.g. media type, resuspension buffer, centrifugation speed, and time). Preparation of single cell suspensions directly from solid tissues or cryopreserved samples may also require additional optimization during dissociation and/or cell handling, which is not covered here.

At this time, methanol fixation of cells has only been validated for compatibility with the standard throughput Single Cell Gene Expression Solution. It has not been validated with the Single Cell Immune Profiling Solution and is not supported. Compatibility with Single Cell Gene Expression HT is untested and thus not recommended at this time.
This demonstrated protocol is optimized for use with standard throughput 10x assays. If using the Single Cell 3’ LT v 3.1 (low throughput) application, diluting cells to the LT specific optimal loading concentrations of 100-600 cells/µl using the resuspension buffer/media can impact assay performance.