Flow Cytometry Protocol

Protocol for Flow Cytometry

Sample Preparation.

  1. Grow cells to 50-75% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
  2. If cells are adherent, harvest gently by washing once with staining buffer and then scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase or Collagenase for a less damaging option.
  3. Reserve 100 μL for counting, then transfer cell volume into a 50 mL conical tube and centrifuge for 8 minutes at 400 RCF.
    1. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting the flow protocol. If cells appear blue, do not proceed.
  4. Re-suspend cells to a concentration of 1 x 106 cells/mL in staining buffer (NBP2-26247).
  5. Aliquot out 1 mL samples in accordance with your experimental samples.


Tip:When cell surface and intracellular staining are required in the same sample, it is advisable that the cell surface staining be performed first since the fixation and permeablization steps might reduce the availability of surface antigens.

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

  1. Recommended: Block non-specific interactions using 0.5-1 µg of a species specific Fc-blocking reagent such as an anti-mouse CD16/CD32 antibody (NBP1-27946).
  2. Add appropriate amount of each directly conjugated antibody (eg. 1 test or 1 µg per sample, as experimentally determined) to 100 µL of staining buffer (NBP2-26247) per sample (eg. use 1 mL of staining buffer for 10 samples).
    1. To determine which directly conjugated antibodies work with your instrument use our panel builder at www.novusbio.com/novusknowsflow.html.
    2. For additional help on selection of conjugated targets, feel free to contact technical support at technical@novusbio.com
  3. Add 100 µL of diluted antibody to each sample.
  4. Incubate on ice (2-8°C) in dark for 1 hour.
  5. Add 1-2 mL of staining buffer and centrifuge at 400 RCF for 5 minutes and discard supernatant.
  6. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 µL for wells) and centrifuging at 400 RCF for 5 minutes.  Discard supernatant.
  7. Resuspend in an appropriate volume of staining buffer (usually 500 µL per sample) 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.

More Resources:

Flow Cytometry (FACS) Illustrated Assay