Flow Cytometry

Sample prep guidelines for cell sorting and analysis

The purpose of this document is to provide general guidelines and best practices for preparing samples for cell sorting. Be mindful that every sample type is different and modifications may be needed.

Cell viability, autofluorescence, and cell aggregation may all affect the overall quality of live cell sorting experiments. Superior cell preparation is crucial and will result in better sort purity, yield, and post-sort cellular function and viability. Please consider the following guidelines for cell preparations intended for cell sorting.

A successful sort results in good purity and yield of the target population and is dependent on these five things:

  1. Cell harvesting and preparation
  2. Cell staining and fluorochrome choice
  3. Proper controls
  4. Sample and sorting conditions
  5. Sample collection conditions

 Buffer Suggestions

  • Use Ca++/Mg++-free buffers. PBS without Ca/Mg is advised. This helps to reduce cell aggregation.
  • Include 0.1-1% BSA or dialyzed FBS at 1-5%. Use a minimal amount of BSA to decrease autofluorescence and to increase population resolution.
  • Avoid non-dialyzed FBS, as it facilitates cell-cell adhesion by replacing Ca and Mg. Add EDTA at 2-5mM to help prevent cell adhesion.
  • High pressure during sorting compromises buffer capacity. Add 10-25mM HEPES to improve pH stability.
  • To samples with reduced cell viability, omit EDTA and add 25-50 ug/mL DNAseI with 5mM MgCl2. This digests free DNA released by dead cells.

Single Cell Suspension

Filter immediately before sorting. Recommended filter is the Celltrics 30um filter (P/N: 04-004-2326). Avoid keeping cells at unnecessarily high concentration. Keep the cell suspension at 10 million/mL during processing, depending on cell type. If there are fewer than 5 million cells in a sample, resuspend in 300- 500uL.

Dead Cell Discrimination

We strongly suggest using a dead cell exclusion dye with any cell sorting experiment. This will greatly reduce autofluorescence and lower non-specific baselines, which will ultimately result in increased population resolution. There is a good selection of dyes for live samples, which will not affect cell physiology for post-sort functional assays.

Cell Staining and Fluorochrome Choice

If there are any questions about fluorochrome choice or cell staining procedures, please consult our staff.

Fluorochrome Selection: Consider doing a preliminary analysis of your experiment before bringing your cells for sorting. Generally, “positive” populations that are dim and only minimally separated from a slightly dimmer "negative" population will lead to a poor analysis and/or a poor sort. A thoughtful balance of fluorochrome brightness with cellular marker abundance is important for optimal resolution of cell populations

Spectral Overlap: Another important consideration in panel design is the amount of spectral overlap between fluorochromes. Spectral spill-over from a very brightly stained channel into a detector that requires high-sensitivity can be a real problem.

Blocking Non-specific Binding: An ideal antibody would have a high affinity to only one, specific cellular epitope. Unfortunately, non-specific binding can be a problem even when using a correctly-titrated antibody. In these cases, a blocking reagent is needed. Usually, a blocking reagent contains a high concentration of species-specific immunoglobulin that can bind to the Fc-receptors that are often responsible for the non-specific binding of the staining antibody.

Titrating Antibodies:

The optimal concentration for antibody labeling is when the ratio of antibody to antigen reaches a point of saturation. Too low and there will not be enough antibody to saturate all of the high affinity binding sites; this will limit brightness, make small pipetting errors significant and make quantitative conclusions about cytometric data suspect. Too high an antibody concentration will waste reagent, and could actually lower the signal-to-noise ratio due to high non-specific binding to low-affinity sites. Antibody titration can reveal the proper range where small changes in antibody concentration or cell number have little effect, but non-specific binding is minimized.

Controls

Compensation Controls: If using cells for single color compensation controls, bring unstained cells as the negative control for compensation. If using beads for compensation, include unstained beads for compensation (as an internal population or a separate tube) and bring unstained cells for an additional technical control.

Gating Controls: Provide proper gating controls. This is essential even for single-color sorts.

Fluorescence Minus One Control: A fluorescence minus one (FMO) control contains all fluorochromes of the multicolor cocktail except one. They represent the combinatorial background fluorescence from other channels into the channel of interest and aid in setting sort gates properly. Many researchers are resistant to using FMO controls on a routine basis because they consume cells, reagents and time, but in many cases they are needed to validate the gating strategy of a reagent panel particularly if you are using the panel for the first time.

Biological Controls: Additional biological controls may be necessary for proper gating, including:

  • Negative Control
  • Positive Control
  • Mock Transfected
  • Treated
  • Untreated

Sorting Formats

Tubes

  • Microfuge tubes (1-4 populations)
  • 12x75mm 5mL tubes (1-4 populations)
  • 15mL tubes (1-2 populations)

Multi-well plates (1 population)

  • 6, 12, 24, 48, 96

Sample Delivery

Samples must be contained in leak-proof tubes, and transported in a leakproof container and a sealed cooler. Cells are typically sorted at approximately 10 million per mL, depending on cell type. 10 million cells takes 1 hour of sort time.  If there are fewer than 5 million cells in a sample, resuspend in 300- 500uL. Bring extra sample buffer (5-15mL), FBS, collection buffer, and collection tubes as backups. Provide proper collection tubes for your application (e.g. sterile, RNAsefree). Bring extra ice for long sorts, if necessary.