Flow Cytometry Experimental Steps for Methanol Permeabilization & Triton X-100 Permeabilization

posted this at Jul 9, 2025 Updated at Jul 9, 2025

Product Manager: Harrison Michael

Important Note: Please refer to the product-specific flow cytometry protocol on the product webpage for corresponding fixation and permeabilization conditions, as well as recommended antibody dilution ratios.

I. Methanol Permeabilization Protocol

A. Solutions and Reagents

Note: Prepare solutions using reverse osmosis deionized water (RODI) or water of equivalent quality.

1. 1× Phosphate Buffered Saline (PBS): To prepare 1 L of 1× PBS, add 100 mL of 10× PBS (P492453) to 900 mL of water and mix.

2. 4% Paraformaldehyde, methanol-free (P395744)

3. 100% Methanol (M116128): Cool before use

4. Antibody Dilution Buffer: Prepare 100 mL of 1× PBS containing 2% FBS by dissolving 2 mL of fetal bovine serum (FBS) in 100 mL of 1× PBS. Store at 4℃.

5. Recommended Secondary Antibodies: To detect uncoupled primary antibodies, select secondary antibodies matching the host species of your primary antibody (e.g., mouse). Click here for the latest list of secondary antibodies approved for flow cytometry.

6. Red Blood Cell Lysis Buffer (R767015)

Note: When adding fluorescent cell dyes (including viability dyes, DNA dyes, etc.) to your experiment, refer to the dye product page for recommended protocols.

B. Fixation

1. Before fixation, detach adherent cells or tissues to form a single-cell suspension.

2. Optimal centrifugation conditions vary by cell type and reagent volume. Generally, 5 minutes at 400–500g is sufficient to pellet cells.

3. If using whole blood, lyse red blood cells and wash by centrifugation before fixation.

4. If epitopes are disrupted by paraformaldehyde and/or methanol, add antibodies targeting CD markers or other extracellular proteins before fixation. These antibodies will remain bound to the target during fixation and permeabilization. Note that certain fluorophores (including PE and APC) are damaged by methanol and should not be added before permeabilization. If unsure, perform a small-scale experiment.

Fixation Procedure:

After centrifugation, resuspend cells in ~100 µL of 4% paraformaldehyde per 1 million cells and fix at room temperature for 10–15 minutes. Alternatively, fix with pre-chilled ice-cold methanol (100%) at -20°C for 5 minutes (methanol fixes and permeabilizes simultaneously, eliminating the need for subsequent permeabilization). After fixation, add an appropriate volume of flow buffer, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant. Resuspend in flow buffer, centrifuge again, and discard the supernatant.

1. Proceed to cellular immunostaining (Section C) or resuspend cells in PBS at 4°C after washing out methanol.

2. Add blocking agent: For human samples, use human serum as an Fc blocking agent; for mouse samples, use 1 µg/mL anti-mouse CD16/CD32 (Ab093991) as an Fc blocking agent.

C. Immunostaining

Note: Count cells using a hemocytometer or alternative method.

1. Aliquot the desired number of cells into tubes or wells (typically 2×10⁵ to 1×10⁶ cells per test).

2. Wash by centrifugation with excess 1× PBS to remove methanol. Discard the supernatant into an appropriate waste container, repeating if necessary.

3. Resuspend cells in 100 µL of diluted primary antibody, prepared in antibody dilution buffer at the recommended dilution or as determined by titration.

4. Incubate at 4°C for 1 hour.

5. Wash by centrifugation in antibody dilution buffer or 1× PBS. Discard the supernatant and repeat washing twice. If using directly conjugated antibodies, skip to step 9.

6. Resuspend cells in 100 µL of diluted fluorescently conjugated secondary antibody (prepared in antibody dilution buffer at the recommended dilution).

7. Incubate at 4°C for 30 minutes.

8. Wash by centrifugation in antibody dilution buffer or 1× PBS. Discard the supernatant and repeat washing twice.

9. Resuspend cells in 200–500 µL of 1× PBS and analyze on a flow cytometer.

Preparation Instructions for Different Samples:

Blood Samples:

1. Place 4 mL of blood in a 50 mL centrifuge tube.

2. Take an appropriate volume of fresh blood and add the corresponding volume of red blood cell lysis buffer.

3. Place on a rotator until the blood becomes translucent, ideally within 10 minutes, and check periodically during lysis.

4. Repeat step 3 once.

5. Resuspend cells in 2% FBS/DPBS at the desired volume

Platelets:

Method 1:

1. Within 10 minutes of blood collection, pipette 100 μL of unstimulated or activated whole blood into a 12 × 75 mm tube containing 1 mL of cold (2–8°C) 4% paraformaldehyde solution.
Note: 100 μL of whole blood yields sufficient fixed blood for 20 tests.

2. Fix platelets at 2–8°C for at least 2 hours. Fixed platelets remain stable for 5 days; store at 2–8°C.

3. Before staining, centrifuge fixed blood at 600 × g for 5 minutes at room temperature (20–25°C).

4. Remove the supernatant and add 1 mL of room-temperature wash buffer (1× PBS).

5. Resuspend the pellet by vortexing, then centrifuge at 600 × g for 5 minutes at room temperature.

6. Remove the supernatant and resuspend the pellet in 1 mL of room-temperature staining buffer.

7. Add ~10 mL of room-temperature 1× red blood cell lysis buffer to 1 mL of whole blood and gently invert.

8. Incubate at room temperature for ~10 minutes, monitoring red blood cell lysis periodically.

9. After lysis, centrifuge at 600 × g for 5 minutes at room temperature (20–25°C).

10. Resuspend cells in 1 mL of FACS buffer, and add 20 μL of cell suspension per well.

11. Add 100 μL of diluted antibody per well, incubate at room temperature in the dark for 30 minutes.

12. After incubation, wash twice with 100 μL of FACS buffer, centrifuging at 600 × g for 5 minutes each time at room temperature.

13. Add diluted secondary antibody.

14. After incubation, wash twice with 100 μL of FACS buffer, centrifuging at 600 × g for 5 minutes each time at room temperature.

15. Resuspend and analyze by flow cytometry.

Method 2:

1. Add 6 μL of whole blood per test to the diluted antibody, mix, and incubate at room temperature in the dark for 40 minutes.

2. Centrifuge at 1200g for 5 minutes, discard the supernatant, and wash once with 200 μL of FACS buffer.

3. Resuspend in 200 μL of FACS buffer and analyze.

II. Triton X-100 Permeabilization Protocol

A. Solutions and Reagents

Note: Prepare solutions using reverse osmosis deionized water (RODI) or water of equivalent quality.

1. 1× Phosphate Buffered Saline (PBS): To prepare 1 L of 1× PBS, add 100 mL of 10× PBS (P492453) to 900 mL of water and mix.

2. 4% Paraformaldehyde, methanol-free

3. Cell Permeabilization Buffer: Prepare 10 mL by adding 30 µL of Triton X-100 (T109027) to 10 mL of antibody dilution buffer. Store at 4°C.

4. Antibody Dilution Buffer: Prepare 100 mL of 1× PBS containing 2% FBS by dissolving 2 mL of fetal bovine serum (FBS) in 100 mL of 1× PBS. Store at 4°C.

5. Recommended Secondary Antibodies: To detect uncoupled primary antibodies, select secondary antibodies matching the host species of your primary antibody (e.g., rabbit). Click here for the latest list of secondary antibodies approved for flow cytometry.

Note: When adding fluorescent cell dyes (including viability dyes, DNA dyes, etc.) to your experiment, refer to the dye product page for recommended protocols.

B. Fixation and Permeabilization

1. Before fixation, detach adherent cells or tissues to form a single-cell suspension.

2. Optimal centrifugation conditions vary by cell type and reagent volume. Generally, 5 minutes at 400–500g is sufficient to pellet cells.

3. If using whole blood, lyse red blood cells and wash by centrifugation before fixation.

4. If epitopes are disrupted by paraformaldehyde and/or Triton X-100, add antibodies targeting CD markers or other extracellular proteins before fixation. These antibodies will remain bound to the target during fixation and permeabilization. If unsure, perform a small-scale experiment.

Procedure:

1. Pellet cells by centrifugation and remove the supernatant.

2. Resuspend cells in ~100 µL of 4% paraformaldehyde per 1 million cells. Mix to dissociate the pellet and prevent cell cross-linking.

3. Fix at room temperature (20–25°C) for 15 minutes.

4. Wash by centrifugation with excess 1× PBS. Discard the supernatant into an appropriate waste container.

5. Resuspend cells in ~100 μL of cell permeabilization buffer per 1 million cells.

6. Incubate at room temperature for 10 minutes.

7. Proceed to staining or store cells in PBS at -4°C overnight.

8. Add blocking agent: For human samples, use human serum as an Fc blocking agent; for mouse samples, use mouse serum as an Fc blocking agent. Add Fc blocking agent to a final concentration of 5% and incubate at room temperature for 15 minutes. Specifically, when using mouse samples and validating PE-conjugated primary antibodies, use 0.2 μg/mL anti-mouse CD16/CD32 (Ab093991) as the blocking agent.

C. Immunostaining

Note: Count cells using a hemocytometer or alternative method.

1. Aliquot the desired number of cells into tubes or wells (typically 2×10⁵ to 1×10⁶ cells per test).

2. Centrifuge cells and discard the supernatant.

3. Resuspend cells in 100 µL of diluted primary antibody, prepared in antibody dilution buffer at the recommended dilution or as determined by titration.

4. Incubate at 4°C for 1 hour.

5. Wash by centrifugation in antibody dilution buffer or 1× PBS. Discard the supernatant and repeat. If using directly conjugated antibodies, skip to step 9.

6. Resuspend cells in 100 µL of diluted fluorescently conjugated secondary antibody (prepared in antibody dilution buffer at the recommended dilution).

7. Incubate at 4°C for 30 minutes in the dark.

8. Wash by centrifugation in antibody dilution buffer or 1× PBS. Discard the supernatant and repeat.

9. Resuspend cells in 1× PBS and analyze on a flow cytometer.