Flow Cytometry Protocol

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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.

a. 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, the cell surface staining should 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 (e.g. 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).

a. To determine which directly conjugated antibodies work with your instrument, use our panel builder at www.novusbio.com/novusknowsflow.html.

b. For additional help on selection of conjugated targets, 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. Re-suspend 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.

PROTOCOL FOR CYTOPLASMIC TARGETS

Optional: Perform cell surface staining as described in the previous section if needed.

1. Fix the cells by adding 100 µL of fixation solution (such as 4% PFA) to each sample for 10-15 minutes.

2. Permeabilize cells by adding 100 µL of a permeabilization buffer to every 1 x 106 cells present in the sample. Mix well and incubate at room temperature for 15 minutes.

a. For cytoplasmic targets, use a gentle permeabilization solution such as 1X PBS + 0.5% Saponin or 0.5% Tween-20.

b. To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (i.e. 0.1% Tween-20 or 0.1 Saponin).

3. Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer solution to each sample.
 

4. Centrifuge for 5 minutes at 400 RCF.
 

5. Discard supernatant and re-suspend in 1 mL of staining buffer + 0.1% permeabilizer.
 

6. Stain each sample at 1 µL/ 1 x 106 cells of primary antibody or 1-3 µL/ 1 x 106 cells for directly conjugated antibodies. Mix well and incubate on ice for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.
 

7. Following the primary/conjugate incubation, add 2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 5 minutes at 400 RCF.
 

8. Remove supernatant and re-suspend each sample in 2 mL staining buffer +0.1% permeabilizer. Repeat wash for 5 minutes at 400 RCF.
 

9. If using a directly conjugated antibody, after the second wash, re-suspend cell pellet to a final volume of 500 µL per sample and proceed with flow analysis.

PROTOCOL FOR NUCLEAR TARGET (NUCLEAR ENVELOPE AND NUCLEAR MATRIX)

Optional: Perform cell surface staining as described in the previous section and/or the cytoplasmic target staining described above.

1. Fix the cells by adding 100 µL fixation solution (such as 4% PFA) to each sample for 10-15 minutes.

2. Permeabilize cells by adding 100 µL of a permeabiliation buffer to every 1 x 106 cells present in the sample. Mix well and
incubate at room temperature for 15 minutes.

a. For nuclear targets, use a gentle permeabilization solution such as 1X PBS (0.1 -1.0%) Triton X-100 or NP-40.

b. To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (Triton or NP40).

3. Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer solution to each sample.

4. Centrifuge for 5 minutes at 400 RCF.

5. Discard supernatant and re-suspend in 1 mL of staining buffer + 0.1% permeabilizer.

6. Stain each sample at 1 µL/ 1 x 106 cells of primary antibody or 1-3 µL/ 1 x 106 cells for directly conjugated antibodies. Mix well and incubate on ice for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.

7. Following the primary/conjugate incubation, add 2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 5 minutes at 400 RCF.

8. Remove supernatant and re-suspend each sample in 2 mL staining buffer +0.1% permeabilizer. Repeat wash for 5 minutes at 400 RCF.

9. If using a directly conjugated antibody, after the second wash, re-suspend cell pellet to a final volume of 500 µL per sample and proceed with flow analysis.

 

More Resources:

Flow Cytometry (FACS) Illustrated Assay