5 Ingredients to Consider in your FACS Buffer
Little tweaks in the FACS acquiring buffer can significantly improve the quality of the recovered cells during sorting by tackling the concerns of viability, auto-fluorescence, and non-specific staining. Here are 5 ingredients to consider for your FACS buffer:
Use Ca/Mg2+ free PBS
Absence of these ions reduces cation-dependent cell to cell adhesion and prevents clumping. A debris free single cell suspension will have lower auto-fluorescence and flow smoothly through the nozzle.
Supplement with FCS (1-10%) or BSA (0.1-1%)
Serum proteins protect cells from apoptosis, prevent non-specific staining and also prevent cells from sticking to the FACS tubes. However, a high concentration of serum can contribute to auto-fluorescence, therefore the optimum serum level should be pilot tested beforehand.
Include EDTA (0.5-5 mM)
EDTA also prevents cation based cell to cell adhesion and so should be included in the buffer if dealing with sticky and adherent cells like macrophages. However, the concentration of EDTA should be titrated first as high levels can be cytotoxic and/or cytocidal.
Slip in some DNase I (25-50µg/ml)
Dead cells release their contents including DNA into the medium which can lead to cell clumping and related problems. If the sample has a high percentage of dead cells – ideally, the dead cells should be removed, but in case that is not feasible due to low cell numbers – it is advisable to add DNaseI into the FACS buffer.
Add sodium azide (0.1-1%)
Sodium azide – at suitable low concentrations – checks bacterial contamination, prevents photo-bleaching of fluorchromes and blocks antibody shedding. As with the EDTA, the optimum concentration should be pilot tested to avoid cell toxicity and death.
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