Shreffler:JAX Tcell studies

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(T regulatory cell surface staining)
Current revision (13:03, 26 January 2011) (view source)
(Collection of Cell Pellet for RNA Isolation)
 
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==Overview==
==Overview==
-
This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.
+
This is a protocol designed for characterizing the mouse allergen (Mus m1)-specific T cell phenotype during the JAX study.
==Materials==
==Materials==
*On average, 18mL of whole heparinized blood/patient will be given.
*On average, 18mL of whole heparinized blood/patient will be given.
-
*8 x 10<sup>6</sup> (8 million) cells for 7 day culture (for measurement of frequency of PBMC-derived, Mus M1-specific CD4+ CD25+ FoxP3+ T cells)
+
*target to isolate 8 x 10<sup>6</sup> (8 million) cells for 48 hour culture
-
*Sterile 5mL polystyrene round-bottom tubes.
+
-
*Phosphate buffered saline (PBS)
+
-
 
+
*Ficoll Paque Plus [Endotoxin tested], room temperature.
*Ficoll Paque Plus [Endotoxin tested], room temperature.
-
*CFSE (5μM aliquots).
 
*Staining buffer (PBS + 5g BSA/L + 2mM EDTA)
*Staining buffer (PBS + 5g BSA/L + 2mM EDTA)
-
 
*Sterile phosphate buffered saline (PBS), room temperature
*Sterile phosphate buffered saline (PBS), room temperature
-
*AIM V Media (w/HSA). Invitrogen # 12055-091 add  5mL PS (PCN streptomycin), and 5mL Glutamine] (Expires 1 month after opening)
+
*AIM V Media (w/HSA). Invitrogen # 0870112DK (Expires 1 month after opening)
-
*Cluster tubes or Cryovial tubes and racks.
+
*Cluster tubes
-
 
+
*Cryovial tubes  
*Turk Blood Diluting Fluid (RICCA # 8850-16).
*Turk Blood Diluting Fluid (RICCA # 8850-16).
-
*IL-2 (approximately 20 units/μL; 1 μg = 2.4x10<sup>3</sup> units) [To make a 10 μg vial of IL-2, reconstitute in 1mL sterile PBS + 1% human AB serum to get a final concentration of 10 μg/mL. Place in aliquots of 50 μL and freeze at -80°].
 
-
 
-
*mAb cocktail including: CD25-PCy5 (10uL), CD4-PC7 (5uL), CD3-APC7 (5uL), Aqua live/ dead (2μL), CD127-PE (1uL)
 
-
 
*Sterile conical tubes (15mL, 50mL)
*Sterile conical tubes (15mL, 50mL)
*CD3, CD28 expander beads (Invitrogen 111-61D)
*CD3, CD28 expander beads (Invitrogen 111-61D)
-
 
-
*1X Facs Lysis Buffer
 
-
 
*Sterile, graduated transfer pipettes.
*Sterile, graduated transfer pipettes.
-
*Mus M1 (50µl) aliquot tube.
+
*Mus M1 (50μL) aliquot tube.
-
 
+
-
*A solution of 20% DMSO + PBS.
+
-
 
+
*Hemocytometer or disposable slides for the automated counter or manual counter
*Hemocytometer or disposable slides for the automated counter or manual counter
-
*Tetanus Toxoid (stock 2 mg/mL).
 
-
 
-
 
-
 
*50 mL Accuspin tubes, radiation sterilized. (Sigma #A-2055)
*50 mL Accuspin tubes, radiation sterilized. (Sigma #A-2055)
-
*24 well tissue culture plates.
+
*Sterile 5mL polypropylene round-bottom tubes
-
 
+
*β-mercaptoethanol (Sigma #M7522)
-
 
+
*RLT lysis buffer (Qiagen, #79216)
-
|Sterile 5mL polypropylene round-bottom tubes
+
-
 
+
-
 
+
-
*15mL conical tubes.
+
-
*5mL polystyrene tubes
+
=='''Procedure'''==
=='''Procedure'''==
Line 64: Line 40:
# Add up to 30mL of diluted blood to the accuspin tube.
# Add up to 30mL of diluted blood to the accuspin tube.
# Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
# Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
-
# Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50 mL conical tube. (Avoid aspirating the Ficoll.) Add PBS to bring to a minimum of 2x the volume, inverting up and down to mix.
+
# Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50mL conical tube. (Avoid aspirating the Ficoll.)  
 +
# Add PBS to bring to a minimum of 3x the volume, inverting up and down to mix.
# Centrifuge at 500 g for 20 minutes at room temperature (maximum acceleration and deceleration).
# Centrifuge at 500 g for 20 minutes at room temperature (maximum acceleration and deceleration).
-
# Aspirate and discard the supernatant. Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding PBS for a total volume of 1 mL. Set aside a 10 µL aliquot of cells for counting as follows: place the 10 µL of cells into 90 µL of PBS, and add 100 µL of Turk's Solution.  Mix well, and let it sit for 1 minute before counting.
+
# Aspirate and discard the supernatant.  
-
# Add 19 mL of PBS to the cells in 990 µL to make a total volume of ~20 mL and centrifuge at 300 g for 15 minutes at room temperature (maximum acceleration and deceleration).
+
# Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding PBS for a total volume of 1 mL.  
 +
# Set aside a 10 μL aliquot of cells for counting as follows: place the 10 μL of cells into 90 μL of PBS, and add 100 μL of Turks Solution.  Mix well, and let it sit for 1 minute before counting.
 +
# Add 19 mL of AIM-V to the cells in 990 μL to make a total volume of ~20 mL.
 +
# Centrifuge at 300 g for 15 minutes at room temperature (maximum acceleration and deceleration).
# In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined.
# In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined.
-
# Carefully introduce 10 µL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter. Calculate the number of cells, taking into account the dilution factors and sample volume used. Or place 20 µL of the stained cells onto a disposable slide and count using the automated cell counter.  
+
# Carefully introduce 10 μL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter.  
-
# After centrifugation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with PBS at 10 x 10<sup>6</sup>/ml in 15 ml PP conical tube.
+
# Calculate the number of cells, taking into account the dilution factors and sample volume used. Or place 20 μL of the stained cells onto a disposable slide and count using the automated cell counter.  
 +
# After centrifugation is completed, aspirate and discard the supernatant.  
 +
# Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with AIM-V at 4 x 10<sup>6</sup>/ml in 15 ml PP conical tube.
==PBMC Stimulation==
==PBMC Stimulation==
'''(Sterile Technique)'''
'''(Sterile Technique)'''
-
# Remove stimulants from the freezer and thaw.
+
# Remove Musm1 from the freezer and thaw.
-
# Label 24 well plate with specimen ID and date (this is for the 7 days cell culture). Label each well with the appropriate stimulant condition, '''ordered by priority''' (for cases where there are insufficient cells to test all stimulants).
+
# Centrifuge the Musm1 briefly in the small centrifuge.
 +
# Label 5 mL sterile polypropylene tubes with the appropriate stimulant condition, '''ordered by priority''' (for cases where there are insufficient cells to test all stimulants).
## Musm1 (allergen):  @ 200 μg/mL purified Musm1 protein in Aim-V.
## Musm1 (allergen):  @ 200 μg/mL purified Musm1 protein in Aim-V.
-
## AIM-V/IL2 (negative control): AIM-V + IL-2 medium alone.
+
## AIM-V (negative control): AIM-V medium alone.
## Beads (positive control): 1μg/mL anti-CD3/CD28 beads.
## Beads (positive control): 1μg/mL anti-CD3/CD28 beads.
-
## Tetanus:  200 μg/mL tetanus in AIM-V.
 
-
# Make 5 mL of PBS + CFSE (2x solution): add 10 μL of stock CFSE (5 mM) into 5 mL of PBS.
 
-
# Add an equal volume (1:1 dilution) of freshly prepared 10 μM CFSE (in PBS) to the tube of cells.
 
-
# Incubate in 37°C water bath for 10 minutes.
 
-
# After incubation, wash the CFSE stained cells in 10 mL of AIM-V @ 300g for 10 minutes at RT.  Aspirate supernatant after centrifugation.
 
-
# Resuspend CFSE stained cells in AIM-V medium @ 4 x 10<sup>6</sup> cells/mL (cell suspension).  For plating, each well should contain 2 x 10<sup>6</sup> cells.
 
-
# Prepare AIM-V + 2X IL-2 solution (AIM-V/IL2) by adding 10 μL of IL-2 to 5 mL of medium in a 15 mL conical tube.  Vortex gently.
 
# Prepare solutions for each stimulant condition as follows:
# Prepare solutions for each stimulant condition as follows:
-
#* 200 μg/mL MusM1 in AIM-V/IL-2: place 450 μL of AIM-V/IL-2 in the appropriately labeled tube and add 50 μL of allergen (MusM1). Then add 500 μL of cell suspension. Pipette up and down, and plate.
+
#* 10 μg/mL MusM1 in AIM-V:  
-
#* AIM-V/IL-2 medium alone: place 500 μL of AIM-V/IL-2 in the appropriately labeled tube and add 500 μL of cell suspension. Pipette up and down and plate.
+
#** place 450 μL of AIM-V in the appropriately labeled tube
-
#* 1 μg/mL anti-CD3, anti-CD28 beads in AIM-V/IL-2: place 500 μL of AIM-V in the appropriately labeled tube and add 2.5 μL of CD3, CD28 expander beads. Then add 500 μL of cell suspension. Pipette up and down and plate.
+
#** add 50 μL of allergen (MusM1)
-
#* 200 μg/mL tetanus in AIM-V/IL-2: place 475 μL of AIM-V/IL-2 in the appropriately labeled tube and add 25 μL of tetanus. Then add 500 μL of cell suspension. Pipette up and down and plate.
+
#** add 500 μL of cell suspension  
-
#* Place the tissue culture plate in the 37°C CO<sub>2</sub> incubator for 7 days.
+
#** pipette up and down to mix
 +
#* AIM-V medium alone:  
 +
#** place 500 μL of AIM-V in the appropriately labeled tube
 +
#** add 500 μL of cell suspension
 +
#** pipette up and down to mix
 +
#* anti-CD3, anti-CD28 beads in AIM-V:  
 +
#** place 500 μL of AIM-V in the appropriately labeled tube
 +
#** add 2.5 μL of CD3, CD28 expander beads
 +
#** add 500 μL of cell suspension
 +
#** pipette up and down to mix
 +
#* Place the culture tubes '''with tube caps loose''' in the 37°C CO<sub>2</sub> incubator for 48 hours.
==Collection of Supernatants==  
==Collection of Supernatants==  
'''(non-sterile technique)'''
'''(non-sterile technique)'''
-
# Cell culture supernatants will be collected for cytokine measurement for 7 day cultures.
+
Note: Cell culture supernatants will be collected for cytokine measurement after 48 hour culture.
-
# At day 7, collect cells carefully by pipetting up and down to resuspend cells in the well, and transfer the total volume in each well into separate 5ml polystyrene tubes. Rinse each well with 200μl of staining buffer and add to corresponding PS tubes. Tap gently to mix.
+
# Visually inspect the cell cultures and make a note of variations in pellet size, culture media color, signs of contamination.
-
# Centrifuge tubes at 300g for 5 minutes at 25°C temperature.
+
# Mix the cells carefully by pipetting up and down to resuspend cells in the tube and evenly mix any secreted cytokine.
 +
# Take a 10 μL aliquot of each culture to inspect under the microscope.
 +
# Mix cell aliquot with equal volume of Turks solution and load 10 μL on a hemacytometer.
 +
# Note variations in cell density, obvious contamination.
 +
# Centrifuge tubes at 400g for 5 minutes at 25°C.
# Obtain a cluster tube rack for the storage of supernatants.
# Obtain a cluster tube rack for the storage of supernatants.
-
# Label cluster tubes as follows:
+
#: Note: There will be two aliquots of supernatant collected for each culture. These should be stored in two separate cluster tube boxes and shipped on separate days to minimize the risk of loss during storage/ shipment.
 +
# Label two cluster tubes for each culture tube as follows:
## Specimen ID
## Specimen ID
## Date
## Date
-
## Supernatants - 7 day
+
## Supernatants - 48 hour
-
## Stimulant condition 1-4
+
## Stimulant condition 1-3
### Stimulant 1 = MusM1  
### Stimulant 1 = MusM1  
### Stimulant 2 = AIM-V
### Stimulant 2 = AIM-V
### Stimulant 3 = Beads
### Stimulant 3 = Beads
-
### Stimulant 4 = Tetanus Toxoid
+
# For each stimulant, using a 1000μL pipette tip, transfer 380 μL of supernatant from the culture tube into each corresponding cluster tube in the cluster rack.  
-
#: For each stimulant, using a 1000μL pipette tip, transfer 800μL of supernatant from the PS culture tube into each corresponding cluster tube in the cluster rack. Be careful not to disturb the cell pellets.
+
#: Take care to not disturb the cell pellets.
-
# Cap the cluster tubes and store in the –80°C freezer (by the freight elevators).
+
# Cap the cluster tubes and store in the –80°C freezer.
-
==T regulatory cell surface staining==  
+
==Collection of Cell Pellet for RNA Isolation==  
'''(non-sterile technique)'''
'''(non-sterile technique)'''
-
# Add 1mL of staining buffer to each tube and vortex.
+
# Prepare RLT buffer containing β-mercaptoethanol by adding 10 μL of β-mercaptoethanol stock solution per 1ml of the RLT buffer.
-
# Centrifuge cells at 300 g for 10 minutes at 4°C. Decant supernatant.
+
#: Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil
-
# Prepare 200 μL mAb cocktail in eppendorf tube:
+
# Add 1 mL of staining buffer to the cell pellet of each culture tube, vortex on low.
-
#* CD25-PCy5 (40 μL)
+
# Centrifuge cells at 400 g for 5 minutes at RT. Please take special care to minimize disturbing the cell pellet.  
-
#* CD4-PC7 (20 μL)
+
# Aspirate supernatant down to line at tube curvature (~50 μL ) using a pasteur pipette with unfiltered 200 μL pipette tip.
-
#* CD3-APC7 (20 μL)
+
# Add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully.  
-
#* Aqua live/ dead (8 μL)
+
# Vortex each tube twice for 15 seconds each time.
-
#* CD127-PE (4 μL)
+
# Transfer to labeled cryovial tube.
-
#* 110 μL staining buffer
+
# Freeze immediately and store for shipment.
-
#: Vortex. Store in fridge (light-sensitive) until ready for use]
+
# Ship to MGH on dry ice.
-
# Add 45 μL of the cocktail to each tube.
+
-
# Incubate @ 4°C for 20-30 minutes.
+
-
# Add ~3mL of staining buffer to each tube and vortex.
+
-
# Centrifuge at 300 g for 10 minutes at 4°C. Decant supernatant. Resuspend cells.
+
-
# To fix/freeze the cells, pipette 500μl of 1X Facs Lysis Buffer into each tube and incubate @ 25°C in the dark for 15 minutes.
+
-
# Prepare a solution of 20% DMSO in PBS
+
-
#: Add 440 μL DMSO to 1.8 mL PBS. Vortex.
+
-
#After the incubation, gradually pipette 500μl of the 20% DMSO + PBS solution into each tube.  
+
-
# Transfer the 1ml of cells + 1X Facs Lysis Buffer + 20% DMSO/PBS solution into an appropriately labeled cluster tubes, and freeze at -80°C for shipment to MGH.
+
==Notes==
==Notes==

Current revision

Contents

Overview

This is a protocol designed for characterizing the mouse allergen (Mus m1)-specific T cell phenotype during the JAX study.

Materials

  • On average, 18mL of whole heparinized blood/patient will be given.
  • target to isolate 8 x 106 (8 million) cells for 48 hour culture
  • Ficoll Paque Plus [Endotoxin tested], room temperature.
  • Staining buffer (PBS + 5g BSA/L + 2mM EDTA)
  • Sterile phosphate buffered saline (PBS), room temperature
  • AIM V Media (w/HSA). Invitrogen # 0870112DK (Expires 1 month after opening)
  • Cluster tubes
  • Cryovial tubes
  • Turk Blood Diluting Fluid (RICCA # 8850-16).
  • Sterile conical tubes (15mL, 50mL)
  • CD3, CD28 expander beads (Invitrogen 111-61D)
  • Sterile, graduated transfer pipettes.
  • Mus M1 (50μL) aliquot tube.
  • Hemocytometer or disposable slides for the automated counter or manual counter
  • 50 mL Accuspin tubes, radiation sterilized. (Sigma #A-2055)
  • Sterile 5mL polypropylene round-bottom tubes
  • β-mercaptoethanol (Sigma #M7522)
  • RLT lysis buffer (Qiagen, #79216)

Procedure

Isolation of Mononuclear Cells

(Sterile Technique)

This is a sterile procedure and all steps should be performed in a hood.

  1. Turn on the hood. Bring Ficoll and PBS to 20°C in the hood.
  2. Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and record subject information, i.e. ID #, date collected, date received.
  3. If performing Basophil Activation assay on this sample, set aside 3mL of whole blood.
  4. Place 15 mL of Ficoll in a 50 mL accuspin conical tube. Centrifuge at 800 g for 1 minute.
  5. Dilute remaining blood at 1:1 with PBS in 50 mL conical tubes.
  6. Add up to 30mL of diluted blood to the accuspin tube.
  7. Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
  8. Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50mL conical tube. (Avoid aspirating the Ficoll.)
  9. Add PBS to bring to a minimum of 3x the volume, inverting up and down to mix.
  10. Centrifuge at 500 g for 20 minutes at room temperature (maximum acceleration and deceleration).
  11. Aspirate and discard the supernatant.
  12. Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding PBS for a total volume of 1 mL.
  13. Set aside a 10 μL aliquot of cells for counting as follows: place the 10 μL of cells into 90 μL of PBS, and add 100 μL of Turks Solution. Mix well, and let it sit for 1 minute before counting.
  14. Add 19 mL of AIM-V to the cells in 990 μL to make a total volume of ~20 mL.
  15. Centrifuge at 300 g for 15 minutes at room temperature (maximum acceleration and deceleration).
  16. In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined.
  17. Carefully introduce 10 μL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter.
  18. Calculate the number of cells, taking into account the dilution factors and sample volume used. Or place 20 μL of the stained cells onto a disposable slide and count using the automated cell counter.
  19. After centrifugation is completed, aspirate and discard the supernatant.
  20. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with AIM-V at 4 x 106/ml in 15 ml PP conical tube.

PBMC Stimulation

(Sterile Technique)

  1. Remove Musm1 from the freezer and thaw.
  2. Centrifuge the Musm1 briefly in the small centrifuge.
  3. Label 5 mL sterile polypropylene tubes with the appropriate stimulant condition, ordered by priority (for cases where there are insufficient cells to test all stimulants).
    1. Musm1 (allergen): @ 200 μg/mL purified Musm1 protein in Aim-V.
    2. AIM-V (negative control): AIM-V medium alone.
    3. Beads (positive control): 1μg/mL anti-CD3/CD28 beads.
  4. Prepare solutions for each stimulant condition as follows:
    • 10 μg/mL MusM1 in AIM-V:
      • place 450 μL of AIM-V in the appropriately labeled tube
      • add 50 μL of allergen (MusM1)
      • add 500 μL of cell suspension
      • pipette up and down to mix
    • AIM-V medium alone:
      • place 500 μL of AIM-V in the appropriately labeled tube
      • add 500 μL of cell suspension
      • pipette up and down to mix
    • anti-CD3, anti-CD28 beads in AIM-V:
      • place 500 μL of AIM-V in the appropriately labeled tube
      • add 2.5 μL of CD3, CD28 expander beads
      • add 500 μL of cell suspension
      • pipette up and down to mix
    • Place the culture tubes with tube caps loose in the 37°C CO2 incubator for 48 hours.

Collection of Supernatants

(non-sterile technique)

Note: Cell culture supernatants will be collected for cytokine measurement after 48 hour culture.

  1. Visually inspect the cell cultures and make a note of variations in pellet size, culture media color, signs of contamination.
  2. Mix the cells carefully by pipetting up and down to resuspend cells in the tube and evenly mix any secreted cytokine.
  3. Take a 10 μL aliquot of each culture to inspect under the microscope.
  4. Mix cell aliquot with equal volume of Turks solution and load 10 μL on a hemacytometer.
  5. Note variations in cell density, obvious contamination.
  6. Centrifuge tubes at 400g for 5 minutes at 25°C.
  7. Obtain a cluster tube rack for the storage of supernatants.
    Note: There will be two aliquots of supernatant collected for each culture. These should be stored in two separate cluster tube boxes and shipped on separate days to minimize the risk of loss during storage/ shipment.
  8. Label two cluster tubes for each culture tube as follows:
    1. Specimen ID
    2. Date
    3. Supernatants - 48 hour
    4. Stimulant condition 1-3
      1. Stimulant 1 = MusM1
      2. Stimulant 2 = AIM-V
      3. Stimulant 3 = Beads
  9. For each stimulant, using a 1000μL pipette tip, transfer 380 μL of supernatant from the culture tube into each corresponding cluster tube in the cluster rack.
    Take care to not disturb the cell pellets.
  10. Cap the cluster tubes and store in the –80°C freezer.

Collection of Cell Pellet for RNA Isolation

(non-sterile technique)

  1. Prepare RLT buffer containing β-mercaptoethanol by adding 10 μL of β-mercaptoethanol stock solution per 1ml of the RLT buffer.
    Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil
  2. Add 1 mL of staining buffer to the cell pellet of each culture tube, vortex on low.
  3. Centrifuge cells at 400 g for 5 minutes at RT. Please take special care to minimize disturbing the cell pellet.
  4. Aspirate supernatant down to line at tube curvature (~50 μL ) using a pasteur pipette with unfiltered 200 μL pipette tip.
  5. Add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully.
  6. Vortex each tube twice for 15 seconds each time.
  7. Transfer to labeled cryovial tube.
  8. Freeze immediately and store for shipment.
  9. Ship to MGH on dry ice.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.

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