Griffin:Flow Cytometry prep & labeling

Direct Immunofluorescence Labeling

1) Lyse Red Blood Cells

NOTE: This will prepare enough cells for 10 samples @ 100ml/test.

• Add 1ml of whole blood to 14ml of RT 1X Ammonium Chloride lysing solution.
• Vortex gently and incubate @ RT for 3-5 minutes. Do NOT exceed 5 minutes.
• Centrifuge for 5 minutes, aspirate supernatant, and gently re-suspend pellet in 5ml of cold 1X PBS.
• Centrifuge again for 5 minutes, aspirate supernatant, and gently re-suspend pellet in 1ml of cold 1X PBS.

2) Cell Surface Staining

• Label tubes.
• Add fluorochrome-conjugated antibody to tubes. (Use 1/0.5mg per 10e6 cells.)
• Add 100ml of RBC-lysed blood to tubes.
• Vortex and incubate for 15-30 minutes in a covered ice bucket.
• Add ~1.5ml of wash buffer (1X PBS) to each tube and mix by inversion.
• Centrifuge tubes @ 1000RPM for 5 minutes, aspirate supernatant, and resuspend pellet in 500ml of 1% paraformaldehyde.

3) Acquire and Analyze Data

Alternately, a lyse/wash procedure can be used in which whole blood is stained, then treated with a “Flow Lysis Buffer” and washed. This “Flow Lysis Buffer” is different from the Ammonium Chloride lysing solution.

Indirect Immunofluorescence Labeling

1) Lyse Red Blood Cells

NOTE: This will prepare enough cells for 10 samples @ 100ml/test.

• Add 1ml of whole blood to 14ml of RT 1X Ammonium Chloride lysing solution.
• Vortex gently and incubate @ RT for 3-5 minutes. Do NOT exceed 5 minutes.
• Centrifuge for 5 minutes, aspirate supernatant, and gently re-suspend pellet in 5ml of cold 1X PBS.
• Centrifuge again for 5 minutes, aspirate supernatant, and gently re-suspend pellet in 1ml of cold 1X PBS.

2) Cell Surface Staining

• Label tubes.
• Add unconjugated primary antibody to tubes. (Use 1-0.5mg per 10e6 cells.)
• Add 100ml of RBC-lysed blood to tubes.
• Vortex and incubate for 15-30 minutes in a covered ice bucket.
• Add ~1.5ml of wash buffer (1X PBS) to each tube and mix by inversion.
• Centrifuge tubes @ 1000RPM for 5 minutes, aspirate supernatant, and re-suspend pellet in 100ml of 1X PBS.
• Add fluorochrome-conjugated secondary antibody to tubes. (Use 1-0.5mg per 10e6 cells.)
• Vortex and incubate for 15-30 minutes in a covered ice bucket.
• Add ~1.5ml of wash buffer (1X PBS) to each tube and mix by inversion.
• Centrifuge tubes @ 1000RPM for 5 minutes, aspirate supernatant, and re-suspend pellet in 500ml of 1% paraformaldehyde.

3. Acquire and Analyze Data

Staining Intracellular Cytokines

1) Activation

Lymphocyte Activation: for 5 samples each (unstim. & act.) @ 100ml/test

                                                             Unstimulated        Activated

RPMI-1640 (+2mM L-glutamine) 500ml 500ml

Brefeldin A 10mg 10mg

PMA -- 25ng

Ionomycin -- 1mg

Whole blood w/ heparin 500ml 500ml

Monocyte Activation: for 10 samples each (unstim. & act.) @ 50ml/test

                                                           Unstimulated        Activated

Brefeldin A 10mg 10mg

LPS -- 1mg

Whole blood w/ heparin 1ml 1ml

Incubate @ 37°C, 7% CO2 for 4 hours.

2) Surface Staining

• Label tubes
• Add fluorochrome-conjugated surface-antigen specific antibodies to tubes. be sure to do pairs: unstimulated and activated.
• Add unstimulated or activated blood to appropriate tubes. Mix well and incubate for 15-30 minutes @ RT in the dark. Keep in mind that some antibodies with a low antigen density may require longer staining times.
• Lyse RBC’s (2ml of Lysing buffer per 100ml of whole blood). Incubate @ RT in the dark for 10 minutes. Do not exceed 15 minutes.
• Centrifuge samples (300-400xg) @ RT, aspirate supernatant, wash once with staining buffer (PBS/Azide + BSA).

3) Fixation and Permeabilization

• Immediately after the wash step, fix the cells by adding 100ml of fixation solution while vortexing the tube and incubate in the dark @ RT for 20 minutes.
• Add 1ml of Permeabilization buffer to each tube, centrifuge for 5 minutes, aspirate supernatant, repeat once.

4) Intracellular Staining

• Dilute the fluorochrome-conjugated anti-cytokine antibody in Permeabilization buffer and add to the appropriate tube. (Use 0.5-0.06mg/10e6 cells.)
• Incubate in the dark @ RT for 20 minutes.
• Add 1ml of Permeabilization buffer to each tube, centrifuge for 5 minutes, aspirate supernatant.
• Resuspend the pellet in 0.5ml staining buffer.

5) Acquire and Analyze Data