Griffin:Flow Cytometry prep & labeling: Difference between revisions
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''Lymphocyte Activation: for 5 samples each (unstim. & act.) @ 100ml/test'' | ''Lymphocyte Activation: for 5 samples each (unstim. & act.) @ 100ml/test'' | ||
Unstimulated/Activated | Component: Unstimulated/Activated | ||
RPMI-1640 (+2mM L-glutamine) | RPMI-1640 (+2mM L-glutamine): 500ml/500ml | ||
Brefeldin A | Brefeldin A: 10mg/10mg | ||
PMA | PMA: --/25ng | ||
Ionomycin | Ionomycin: --/1mg | ||
Whole blood w/ heparin | Whole blood w/ heparin: 500ml/500ml | ||
''Monocyte Activation: for 10 samples each (unstim. & act.) @ 50ml/test'' | ''Monocyte Activation: for 10 samples each (unstim. & act.) @ 50ml/test'' | ||
Unstimulated/Activated | Component: Unstimulated/Activated | ||
Brefeldin A | Brefeldin A: 10mg/10mg | ||
LPS | LPS: --/1mg | ||
Whole blood w/ heparin | Whole blood w/ heparin: 1ml/1ml | ||
Incubate @ 37°C, 7% CO2 for 4 hours. | Incubate @ 37°C, 7% CO2 for 4 hours. |
Revision as of 10:31, 2 October 2008
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
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.
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 and