Salk Institute
Flow Cytometry Core Facility

Analysis

Preparing to run samples at FCCF:

Work Flow

Comments

1. Perform Experiment

Treatment of cells/animals under different conditions

2. Harvest cells

Dissociate tissue/detach cultures/spin down cells

3. Perform any fluorescence labeling

Antibodies/other probes; post staining fixative if needed. Remember to include controls.

5. Prepare cells to run on analyzer

Re-suspend in appropriate buffer and concentration in Falcon brand tubes (352052, 352054 or 352235)


The final step after gently harvesting your cells (for apoptosis studies, remember to spin down and include cells from the culture supernatant) and performing any required fluorescence labeling is to re-suspend them appropriately. The goal is to minimize clumping, and stabilize the samples until data acquisition.

Basic FACS Buffer for re-suspending samples:

 

Component

Purpose

Basic Buffer

Ca2+/Mg2+ free PBS  (1x)

Isotonic buffer, phenol red free

1% BSA, or 2% FBS (Ca2+/Mg2+ free)

Protein stabilizer

+ for sticky cells

2mM EDTA (chelator)

Reduces cation mediated cell adhesion

+ for dead cells

DNAse

*See below for further information


*DNAse: 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  

Sufficiently concentrated samples can be run at the LOWEST flow rate to yield event rates less than the cytometer’s processing limit (LO flow rate yields maximal instrument sensitivity). This will also help minimize acquisition time.

The guideline is up to 0.2 - 1 x 10^6 cells/ml in 300-500ul (no more than 2ml) but depending on your sample (if your sample is sticky or clumpy you will have no choice but to keep it more dilute) and the instrument you are using there are some adjustments that you can make; refer to the table below:

Instrument and Processing Rate

Cell Concentrations

FACScan - Limited by analogue electronics

  • Run cells ≤ 1500 evt/s due for samples to be processed correctly (300 evt/s for DNA in LO)

2 x 10^5 – 1 x 10^6 cells/ml

Dilute cells if running too fast in LO

LSRII – Faster digital processing speeds

  • Capable of processing much greater event rates without data loss; up to **20,000 evt/s.

**CAVEAT: Realistically, this rate is only applicable to clean lymphoid preps concentrated sufficiently! Limit yourself to up to 10K evt/s for non-clumpy adherent cells.

Adherant cells
2 x 10^5 – 1 x 10^6 cells/ml

Clean lymphocytes:
1-2 x 10^7 cells/ml

 

Use a higher volume to enable you to set up comfortably before the sample is all gone, you can make the samples up in a smaller volume. For adherent cells, your sample concentration will need to be more dilute than suspension cells (eg lymphocytes).

Sample Tubes: Only use Falcon brand tubes that are approved. If your sample has any obvious clumps (visually inspect tubes), you will need to filter your cells prior to installing on the instrument to avoid causing a clog, so consider using 352235 (with filter cap).

*Do not neglect to bring compensation tubes and other relevant controls
(isotype/neg/FMO/unstimulated/healthy…. etc )


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