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, minimizing cell suspension volume added to the Single Cell Master Mix is recommended. 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 load the final resuspension in an approved buffer or media.
Note for Feature Barcode technology:
Most buffers and media discussed in the Cell Preparation Guide were not specifically tested with Cell Surface Protein labeling; however, we consider using these alternative buffers with this application to be low-risk. Please see this article for additional guidance regarding buffers tested with CellPlex labeling.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, CellPlex, Fixed RNA Profiling Gene Expression