Salk Institute
Flow Cytometry Core Facility

Sorting

Preparing samples for a sort at FCCF:

A clump free (filter before bringing to FCCF) single cell suspension in Sort Buffer at the acceptable concentration range is required in order avoid clogging the instrument and allow sorting to proceed.

Many variables can affect your sample (e.g. cell harvesting protocol, cell concentration for sorting, sorting buffer, collection media, collection temperature, sorter set up). Please note that it may be necessary to spend a little time working with us to identify optimal parameters.

AVOID DELAYS TO YOUR APPOINTMENT: The Flow Cytometry Core Facility at the Salk Institute is committed to providing the highest standard of service for flow cytometry to support researchers in their quest to obtain accurate and meaningful data. Inaccurate fluorescence compensation can give rise to misleading data interpretation and incorrect identification of cell populations. In order to assure consistency and reproducibility, FCCF cannot proceed with running samples until compensation controls requirements are met by the user.

Work Flow

Comments

1. Make a Request for Sorting

Provide details of your needs and experiment/sample

2. Perform Experiment

Treatment of cells/animals under different conditions

3. Harvest cells

Dissociate tissue/detach cultures/spin down cells

4. Perform any fluorescence labeling 

Antibodies/other probes
Remember to include controls.

5. Prepare for FACS appointment

  • Re-suspend filtered cells in Sort Buffer  (1ml min.)
    • Non-adherent cells (e.g. lymphocytes):
      8-15 million/ml
    • Adherent cells/tissue derived:
      ≤ 5 million/ml
  • Bring cells to FCCF in sterile Falcon brand tubes (352054, or 352235 with cell strainer)
  • Bring appropriate collection vessels with pre-aliquoted Collection Media
  • Bring extra Sort Buffer and Collection Media
  • Ensure you have included compensation controls

 

Basic Sort Buffer for re-suspending samples

Note: Culture media is generally not recommended; as a start, it has poor pH buffering outside of the CO2 incubator. Difficult sample types may require testing of different conditions, but a good place to start is with the basic Sort Buffer:

 

Component

Purpose

Basic Buffer
Sterilize using 0.2uM filter, store at 4°C

Ca2+/Mg2+ free PBS (1x)

Isotonic buffer, phenol red free

25mM HEPES pH 7.0

pH buffer

*1% FBS
(heat inactivated)

Protein stabilizer

+ for adherent cells

**2mM EDTA (chelator)

Reduces cation mediated cell adhesion

+ for dead cells

***DNAse

See below for further information


* Serum can be increased if viability is an issue, up to 5% only; high levels of protein causes stream fanning (compromises sort purity).

** EDTA can be increased to 5mM if needed and viability is not compromised

*** DNA released by dead cells is sticky. Minimize issues by gently harvesting cells (consider Accutase, Accumax instead of trypsin). In cases where cell death is unavoidable (eg fragile cells, or retrieving cells from tissue) it may be beneficial to incorporate DNAse during the harvesting, prep and wash steps in the absence of EDTA as the enzyme requires cations for activity. For particularly problematic samples, add DNAse (~10 U/ml or 1 µg/ml) to the FACS buffer.

Sample Concentration and Volume  

Make sure you count your cells and adhere to the recommended concentrations to avoid time lost due to clogging (non-adherent cells: 8-15 million/ml, adherent cells: up to 5 million/ml; minimum sample volume of 1ml).

Besides clogging the nozzle, overly concentrated samples may have high coincidence aborts (cells arriving too close to be sorted) or cause stream fanning; too dilute samples will lead to prolonged sort times (or compromised instrument detection sensitivity if flow rate is increased). Sticky samples may benefit from being slightly more dilute – optimization usually involves some degree of trial and error for different samples.

Collection Media

For live sorts, a good collection media is pH stable and buffered with high % serum (eg 50%) to keep cells happy; we can also sort into lysis buffer such as Qiagen RLT for RNA (no Trizol or phenol containing solutions). Bring collection vessels with collection media pre-aliquoted.



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