Question: What buffers can be used for washing and cell resuspension?
Answer: To prepare single cells for Chromium Single Cell applications, it is recommended to use 1X PBS (calcium and magnesium-free) containing 0.04% weight/volume BSA (400 µg/ml) for washing and resuspension. Fresh and frozen/thawed PBMC samples and cell lines have been tested with this buffer.
Primary cells, stem cells, and other sensitive cell types may require washing and resuspension in alternative buffers to maximize viability. For other buffers that have been tested, please refer to page 3 of the Cell Preparation Guide. It is also possible to use most cell culture media with up to 10% FBS or up to 2% BSA to maintain cell health with little to no adverse downstream effects.
Media should not contain excessive amounts of EDTA (> 0.1mM), or magnesium (> 3mM) as those components will inhibit the reverse transcription reaction.
Any surfactants (Tween-20, etc.) should also be avoided as they may interfere with GEM generation.
When using untested buffers or cell culture media, it is recommended to minimize cell suspension volume added to the Single Cell Master Mix. This will reduce carryover and potential adverse effects of any buffer components. Many proprietary buffers may contain EDTA or Mg2+; therefore, when preparing a sample using any proprietary buffers, we recommend incorporating a wash step to remove any additives that may inhibit reverse transcription and perform the final resuspension in an approved buffer or media.
Most buffers and media discussed in the Cell Prep guide were not tested for use with Cell Surface Protein labeling; however, we consider using these alternative buffers with this application to be low-risk.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, CellPlex