Useful Tips to Improve Sample Quality for the Flow Cytometric Analysis and Cell Sorting (Live Cells)
Low cellular viability, auto-fluorescence, and cell aggregates will result in poor flow cytometric analysis and sorts. Improved cell preparation will result in better sort purity, yield, and cell viability
- Make all cell preparations strictly on ice, unless otherwise stated in the procedure
- Buffer suggestions
- Use Ca/Mg++ free buffers (for example: DPBS, 1X without calcium & magnesium, Sterile, Cat#21-031-CV, Mediatech, Inc)
- Use BSA (0.1 - 1%) or dialyzed FBS (1-5%) (any source will be good) for the protein support of the buffer. Addition of non-dialyzed serum to the buffers will replace the Ca/Mg++. Use lowest sufficient concentration of BSA to improve data quality (decrease auto-fluorescence and increase population resolution)
- Use EDTA at up to 5mM concentration (for example: ready to use 0.02% in DPBS (0.5 mM), Cat# E8008, Sigma-Aldrich Co.; or ~0.5 M in H2O, Cat#03690, Sigma-Aldrich Co.). It may help to prevent cation-dependent cell-cell adhesion
- Add 25-50ug/ml of DNAse I (Cat# D4513, Sigma-Aldrich Co.) and 5mM MgCl2 (Magnesium chloride hexahydrate, Cat# M2670, Sigma-Aldrich Co.) to the buffer. It may help if cells are clumping due to the cell death
- Single cell suspension
- At any stage syringe the sample through the 25 gauge needle, 3-5 slow passages
- Filter samples just prior to analysis or cell sorting to remove remaining clumps. The 12 x 75-mm tube with the cell-strainer cap ("blue-cap tubes"; #161900, RU Stockroom; or Cat#352235, BD Falcon) offers the convenient way to filter laboratory samples. Note: It's important to prevent clumping (using tips above) before you filter your samples through a nylon mesh, otherwise you will lose too many cells
- Keep cells at reasonable concentration of 1-5-10 million/ml, dependent on the cell type. Avoid keeping cells at unnecessary high concentration for the prolonged periods of time
- Use dead cell exclusion/discrimination dyes to "eliminate" dead cells from analysis and sort. Choice of dead cell exclusion dye depends on the color combination of fluorochromes within the sample
- For non-fixed cells use conventional dead cell exclusion dyes, for example - PI, 7AAD, DAPI, SYTOX Blue, Green, or Red; etc. Note: DO NOT USE conventional dead cell exclusion dyes (listed above) on the fixed cells
- For fixed cells - use fixable dead cell exclusion dyes, for example - LIVE/DEAD Fixable Blue, Aqua, Violet, Green, or Red; etc.
Avoid:
- Vigorous vortexing. It could damage fragile cells
- Excessive and unnecessary centrifuge spin. Use minimal necessary forces (rpm) to sediment cells
- Aspirating the entire buffer, leaving dry pellet of cells. Cells will die in the dry pellet
- Bubbles. In bubbles due to the surface tension forces cells tend to die
