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Guidelines for flow cell analysis or cell sorting

Flow Analysis

• Generally, 0.3 -0.4 million cells should suffice if 10,000 events need to be acquired

• Please resuspend cells in a minimum volume of approximately 400 µl and filter them prior to analysis

• Load samples using 5 ml polystyrene FACS tubes as the polypropylene ones are unable to fit on the analyser

Please prepare appropriate compensation controls, gating controls and add a viability dye when analysing live populations.

Flow Cell Sorting

• As a general rule, cell lines can be resuspended at a concentration of 5 million cells/ml while PBMCs or BM samples can be resuspended at approximately 10 million cells/ml.

• Please aliquot samples into 5 ml polypropylene tubes (opaque tubes) to minimize cell loss.

• Sample CAN be resuspended in phenol-red free media or calcium magnesium free PBS containing 2-3% FBS (with or without 2 – 5 mM EDTA)

• Dead cells can stain non-specifically. It is ideal to add a viability dye (e.g. DAPI, PI, 7AAD etc) to enhance recovery of live cells during the sort.

• Please filter your samples if possible* to prevent clogs. A clog to the machine may compromise the purity of your sort. Adding DNAse (samples with high cell death) or EDTA to your sample can help reduce clumping.

• Please be aware of the maximum flow rate of the sorter that is suitable for your cells. Generally, flow rate 3 – 5 gives best results for live cell sorting.  It would take approximately 30 – 45 minutes for 1mL of sample to be sorted, depending on complexity of sort, rarity of population, viability and cell concentration.

• Prepare appropriate and accurate controls for compensation and gating

• While setting up voltages, always leave at least a half log difference between the positive peaks of fluorophores to maximise chances of your compensation succeeding.