Flow Cytometry Core

Services

Salk Institute for Biological Studies - Flow Cytometry Core - Services

Services



Using the Flow Cytometry Core Facility

The following is basic information to help new users understand how to use the various FCCF services. Please also read our safety notices.

Getting started at FCCF: Create yourself a *PPMS account; users of the Biophotonics Core already have a PPMS account set up:

  • Scheduling and Rates → Account Request

You will need to provide your Fund #, for recharging purposes – ask your supervisor/lab manager for this. Please note also that resource schedules are only activated after your access has been approved following training with FCCF.

For analysis, bring samples ready to run at FCCF in Falcon 2052 tubes. Samples for sorts: use sterile capped tubes eg. 352003, or 352235 (filter tops) for convenience.

Analytical Flow Cytometry Instrumentation

FCCF has bench-top analyzers available for independent operation. Contact the facility for advice on experimental design and instrument selection.

New users: If you are new to the technology, review the short introductory courses listed below before contacting FCCF for instrument training:

Request training via PPMS well before setting up an experiment as the cytometers have different capabilities; we want to verify your fluorochrome choices are compatible as well as ensure you have optimal experimental design before money is spent on reagents (i.e. fluor/probe selections and combinations).

Cytometer

Training

Materials

FACScan (1- laser, 3 color)

1-2hr

Small number of relevant experimental samples (discuss your experiment first with facility staff)

LSR II (5 laser, 15 color)

2 hours

Beads will be provided for training.

Post training support: Keep your first few experiments simple while you are learning to run an experiment unassisted, and please coordinate with us during this initial phase so we can support you if needed – we want your experiments to be successful. 

Fluorescence Activated Cell Sorting (FACS)

The Flow Cytometry Core Facility has a multi-laser Becton Dickinson FACS Vantage SE DiVA cell-sorter, which is operated by staff. Depending on your needs and scheduling availability, it may take up to 1 week to accommodate you.

Sort Requests: Contact us well before you set up your experiment so that we may discuss your exact requirements. You may do this through PPMS.

Note: Some cells are more fragile than others, and your samples are important to us: we will optimize instrument set up to achieve the best sample recovery for your individual needs.

Sample preparation is critical for the success of your sort and to minimize down time due to a clogged sort nozzle.

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.

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 )