Question: How do I prepare extremely precious or fragile cells?
Answer: If your samples are extremely precious (less than 1x10^5 total cells) or fragile, it is possible to reduce the number of centrifugation and/or washing steps to reduce cell loss. Centrifugation speed may also be reduced to avoid cell damage.
If the number of cells is very small and the suspension is free of debris, it may be possible to skip washing altogether and load the cells directly into the chip in their native media conditions.
For fragile cells, PBS + 0.04% BSA can be replaced with most common cell culture buffers (DPBS, HBSS) or media (EMEM, DMEM, IMEM, RPMI, Ham's F12, DMEM/F12) + 10% FBS or M199 with minimal reduction in performance. If there is a specific buffer formulation/native media that is required for maintaining a single cell suspension or preserving cell integrity, we recommend using it.
However, buffers or media should not contain EDTA or magnesium as those components will inhibit the reverse transcription reaction. If they must be used, it is recommended to wash the cells and transfer the minimal volume of cell suspension into the Master Mix to reduce carryover.
For a cell preparation protocol optimized for limited samples, see Section 3 of the Cell Preparation Guide: https://support.10xgenomics.com/single-cell-gene-expression/index/doc/demonstrated-protocol-single-cell-protocols-cell-preparation-guide
Products: Single Cell 3', VDJ