Griffin:Flow Cytometry prep & labeling

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.

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.

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.

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.

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.

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).

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.

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

Component:                                      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

Component:                                      Unstimulated/Activated


 * Brefeldin A:                                        10mg/10mg
 * LPS:                                                           --/1mg
 * Whole blood w/ heparin:                    1ml/1ml

Incubate @ 37°C, 7% CO2 for 4 hours.