Griffin:Flow Cytometry prep & labeling: Difference between revisions

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(New page: ==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 A...)
 
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==Direct Immunofluorescence Labeling==
==Direct Immunofluorescence Labeling==


'''1)''' Lyse Red Blood Cells
[[Image:Spectra3.jpg]]
 
===Lyse Red Blood Cells===


'''NOTE:'''  This will prepare enough cells for 10 samples @ 100ml/test.
'''NOTE:'''  This will prepare enough cells for 10 samples @ 100ml/test.
Line 10: Line 12:
* Centrifuge again for 5 minutes, aspirate supernatant, and gently re-suspend pellet in 1ml 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
===Cell Surface Staining===


* Label tubes.
* Label tubes.
Line 19: Line 21:
* Centrifuge tubes @ 1000RPM for 5 minutes,  aspirate supernatant, and resuspend pellet in 500ml of 1% paraformaldehyde.
* Centrifuge tubes @ 1000RPM for 5 minutes,  aspirate supernatant, and resuspend pellet in 500ml of 1% paraformaldehyde.


'''3)''' Acquire and Analyze Data
===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.
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.
Line 25: Line 27:
==Indirect Immunofluorescence Labeling==
==Indirect Immunofluorescence Labeling==


'''1)''' Lyse Red Blood Cells
===Lyse Red Blood Cells===


'''NOTE:'''  This will prepare enough cells for 10 samples @ 100ml/test.
'''NOTE:'''  This will prepare enough cells for 10 samples @ 100ml/test.
Line 34: Line 36:
* Centrifuge again for 5 minutes, aspirate supernatant, and gently re-suspend pellet in 1ml 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
===Cell Surface Staining===


* Label tubes.
* Label tubes.
Line 47: Line 49:
* Centrifuge tubes @ 1000RPM for 5 minutes,  aspirate supernatant, and re-suspend pellet in 500ml of 1% paraformaldehyde.
* Centrifuge tubes @ 1000RPM for 5 minutes,  aspirate supernatant, and re-suspend pellet in 500ml of 1% paraformaldehyde.


3. Acquire and Analyze Data
===Acquire and Analyze Data===


==Staining Intracellular Cytokines==
==Staining Intracellular Cytokines==


'''1)''' Activation
===Surface Staining===


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


                                                              Unstimulated        Activated
===Fixation and Permeabilization===


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


Brefeldin A                                                    10mg            10mg
'''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.


PMA                                                                      --            25ng
===Acquire and Analyze Data===


Ionomycin                                                            --              1mg
==Cell Activation==


Whole blood w/ heparin                            500ml            500ml
===Lymphocyte Activation===


''Monocyte Activation:  for 10 samples each (unstim. & act.) @ 50ml/test''
for 5 samples each (unstim. & act.) @ 100ml/test


                                                            Unstimulated       Activated
Component:                                      Unstimulated/Activated


Brefeldin A                                                   10mg             10mg
*RPMI-1640 (+2mM L-glutamine): 500ml/500ml
*Brefeldin A:                                        10mg/10mg
*PMA:                                                      --/25ng
*Ionomycin:                                            --/1mg


LPS                                                                        --              1mg
Whole blood w/ heparin:                  500ml/500ml


Whole blood w/ heparin                                   1ml             1ml
===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.
Incubate @ 37°C, 7% CO2 for 4 hours.


'''2)''' Surface Staining
==Cell Detachment==
 
===EDTA===


* Label tubes
EDTA solution chelates calcium, is less stringent than trypsin, and may help preserve ECM epitopes.
* 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
*10mM EDTA (<15mM EDTA). Pipet EDTA solution into flask/plate and incubate for 1 minute. Gently tap the flask to determine cell adherence. Allow to incubate at 37C up to 10 minutes. Increase EDTA mM concentration according to strength of adherent cell.


* 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.
===Citric Saline===
* Add 1ml of Permeabilization buffer to each tube, centrifuge for 5 minutes, aspirate supernatant, repeat once.


'''4)''' Intracellular Staining
*500mL 10x solution: 50.3 g KCl, 22.06 g Sodium Citrate.
* 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
Pipet prewarmed 1X Citric Saline solution into flask/plate and incubate at 37C for 4 minutes. Remove cells be gently tapping or pipetting up and down several times.

Revision as of 11:51, 22 September 2011

Direct Immunofluorescence 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.

Indirect Immunofluorescence 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 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.

Acquire and Analyze Data

Staining Intracellular Cytokines

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.

Acquire and Analyze Data

Cell 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.

Cell Detachment

EDTA

EDTA solution chelates calcium, is less stringent than trypsin, and may help preserve ECM epitopes.

  • 10mM EDTA (<15mM EDTA). Pipet EDTA solution into flask/plate and incubate for 1 minute. Gently tap the flask to determine cell adherence. Allow to incubate at 37C up to 10 minutes. Increase EDTA mM concentration according to strength of adherent cell.

Citric Saline

  • 500mL 10x solution: 50.3 g KCl, 22.06 g Sodium Citrate.

Pipet prewarmed 1X Citric Saline solution into flask/plate and incubate at 37C for 4 minutes. Remove cells be gently tapping or pipetting up and down several times.

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