Flow Cytometry Protocol

General Flow Cytometry (FACS) Protocol

Prepare your samples.

  1. Determine the number of cells required for staining.  Typically between 2 x 105 and 1 x 106 cells are required for each test sample, although more may be necessary for certain experiments.Remember to harvest enough cells for controls including unstained cells and cells for an isotype control, positive control, known negative control, etc.
  2. Harvest cells (if adherent, scraping is preferred as trypsin can destroy certain epitopes of proteins; if primary cells from tissue samples are being used, prepare a single cell suspension via mechanical disruption or enzymatic digestion (eg. Collagenase) followed by clean up with a nylon mesh cell strainer).  Centrifuge the cells at 400 RCF for 5 minutes and resuspend in 2-3 ml PBS.
  3. Count cells using a hemocytometer.
  4. Prepare aliquots of cells (same range as identified in step 1) into fresh tubes or wells.     
  5. Add additional PBS (1 ml for tubes or 100 ul for wells) and centrifuge at 400 RCF for 5 minutes.
  6. Aspirate supernatant and preform an additional wash by adding fresh PBS (1 ml for tubes or 200 ul for wells) and gently resuspending the cell pellet.  Centrifuge at 400 RCF for 5 minutes and discard supernatant.
  7. Resuspend cells in an appropriate volume of staining buffer (i.e. NBP2-26247).  Transfer to a flow cytometer compatible tube or plate.

Extracellular staining (If proteins or antigens are intracellular, proceed to next section).

  1. Recommended: Block non-specific interactions using 0.5-1.0 ug of a species specific Fc-blocking reagent (e.g. for staining mouse cells you could use anti-mouse CD16/CD32 antibody NBP1-27946).
  2. Add appropriate amount of each directly conjugated antibody (eg. 1 test or 1ug per sample, as experimentally determined) to 100 ul of staining buffer (NBP2-26247) per sample (eg. use 1 ml of staining buffer for 10 samples). To determine which directly conjugated antibodies work with your instrument use our panel builder at www.novusbio.com/novusknowsflow.html.
  3. Add 100 ul of diluted antibody to each sample.
  4. Incubate on ice (2-8C) in the dark for 1 hour.
  5. Centrifuge at 400 RCF for 5 minutes and discard supernatant.
  6. Wash twice by resuspending cells in staining buffer (1 ml for tubes or 200 ul for wells) and centrifuging at 400 RCF for 5 minutes.  Discard supernatant.
  7. Resuspend in an appropriate volume of staining buffer and proceed with analysis on your flow cytometer.

Intracellular Staining.
Tip: When performing intracellular staining, it is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location.  Generally, our Intracellular Flow Assay Kit (NBP2-29450) is a good place to start as it contains an optimized combination of reagents for intracellular staining as well as an inhibitor of intracellular protein transport (necessary if staining secreted proteins).  Certain targets may require more gentle or transient permeabilization protocols such as the commonly employed methanol or saponin-based methods.

  1. Optional: Perform cell surface staining as described in the previous section.
  2. Following final wash from previous step (either extracellular staining or sample preparation), resuspend cells in appropriately diluted fixation/permeabilization buffer (1 ml for tubes or 100 ul for wells).
  3. Pulse with vortex or pipette up and down vigorously and incubate for 30-60 minutes in the dark.
  4. Wash 2 times by adding additional permeabilization buffer (2 ml for tubes or 200 ul for wells) and centrifuging at 400 RCF for 5 minutes.  Discard supernatant.
  5. [Optional] Block by adding 2 ul of appropriate blocking reagent (eg. mouse serum, NBP1-27946 (Fc-block), etc.) to residual volume remaining in the tube or well.  Incubate on ice (2-8C) in the dark for 15 minutes.
  6. Add appropriate amount of each directly conjugated antibody (eg. 1 test or 1ug per sample, as experimentally determined) to 100 ul of appropriately diluted permeabilization buffer per sample (eg. use 1 ml of staining buffer for 10 samples). To determine which directly conjugated antibodies work with your instrument use our panel builder at www.novusbio.com/novusknowsflow.html.
  7. Add 100 ul of diluted antibody cocktail to each sample.
  8. Incubate on ice (or 2-8C) in the dark for 1 hour.
  9. Centrifuge at 400 RCF for 5 minutes and discard supernatant.
  10. Wash 2 times by adding additional permeabilization buffer (2 ml for tubes or 200 ul for wells) and centrifuging at 400 RCF for 5 minutes.  Discard supernatant.
  11. Resuspend in an appropriate volume of staining buffer and proceed with analysis on your flow cytometer.

More Resources:

Flow Cytometry (FACS) Illustrated Assay