Shreffler:JAX Tcell studies: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 73: | Line 73: | ||
'''(non-sterile technique)''' | '''(non-sterile technique)''' | ||
# Cell culture supernatants will be collected for cytokine measurement | # Cell culture supernatants will be collected for cytokine measurement after 48 hour culture. | ||
# | # After 48 hours, mix the cells carefully by pipetting up and down to resuspend cells in the tube to evenly mix any secreted cytokine. | ||
# Centrifuge tubes at 300g for 5 minutes at 25°C | # Centrifuge tubes at 300g 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: | # Label cluster tubes as follows: | ||
## Specimen ID | ## Specimen ID | ||
## Date | ## Date | ||
## Supernatants - | ## Supernatants - 48 hour | ||
## Stimulant condition 1-3 | ## Stimulant condition 1-3 | ||
### Stimulant 1 = MusM1 | ### Stimulant 1 = MusM1 | ||
### Stimulant 2 = AIM-V | ### Stimulant 2 = AIM-V | ||
### Stimulant 3 = Beads | ### Stimulant 3 = Beads | ||
#: For each stimulant, using a 1000μL pipette tip, transfer 800μL of supernatant from the | #: For each stimulant, using a 1000μL pipette tip, transfer 800μL of supernatant from the culture tube into each corresponding cluster tube in the cluster rack. Be careful not to disturb the cell pellets. | ||
# Cap the cluster tubes and store in the –80°C freezer. | # Cap the cluster tubes and store in the –80°C freezer. | ||
Revision as of 10:33, 23 March 2010
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
- Phosphate buffered saline (PBS)
- 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 or Cryovial tubes and racks.
- 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.
- A solution of 20% DMSO + PBS.
- 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)
- 1.5 mL DNase, RNase free Eppendorf tubes (Fisher #05-402-25)
Procedure
Isolation of Mononuclear Cells
(Sterile Technique)
This is a sterile procedure and all steps should be performed in a hood.
- Turn on the hood. Bring Ficoll and PBS to 20°C in the hood.
- Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and record subject information, i.e. ID #, date collected, date received.
- If performing Basophil Activation assay on this sample, set aside 3mL of whole blood.
- Place 15 mL of Ficoll in a 50 mL accuspin conical tube. Centrifuge at 800 g for 1 minute.
- Dilute remaining blood at 1:1 with PBS in 50 mL conical tubes.
- 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).
- 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.
- 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.
- 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).
- 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.
- 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 106/ml in 15 ml PP conical tube.
PBMC Stimulation
(Sterile Technique)
- Remove stimulants from the freezer and thaw.
- Label 5 mL sterile polypropylene tubes, each well 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.
- AIM-V (negative control): AIM-V medium alone.
- Beads (positive control): 1μg/mL anti-CD3/CD28 beads.
- Wash and resuspend freshly isolated PBMCs in AIM-V medium @ 4 x 106 cells/mL (cell suspension). For culture, each tube will contain 2 x 106 cells (0.5 mL).
- Prepare solutions for each stimulant condition as follows:
- 200 μg/mL MusM1 in AIM-V: place 450 μL of AIM-V in the appropriately labeled tube and add 50 μL of allergen (MusM1). Then add 500 μL of cell suspension. Pipette up and down.
- AIM-V medium alone: place 500 μL of AIM-V in the appropriately labeled tube and add 500 μL of cell suspension. Pipette up and down.
- 1 μg/mL anti-CD3, anti-CD28 beads in AIM-V: 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.
- Place the tissue culture plate in the 37°C CO2 incubator for 48 hours.
Collection of Supernatants
(non-sterile technique)
- Cell culture supernatants will be collected for cytokine measurement after 48 hour culture.
- After 48 hours, mix the cells carefully by pipetting up and down to resuspend cells in the tube to evenly mix any secreted cytokine.
- Centrifuge tubes at 300g for 5 minutes at 25°C.
- Obtain a cluster tube rack for the storage of supernatants.
- Label cluster tubes as follows:
- Specimen ID
- Date
- Supernatants - 48 hour
- Stimulant condition 1-3
- Stimulant 1 = MusM1
- Stimulant 2 = AIM-V
- Stimulant 3 = Beads
- For each stimulant, using a 1000μL pipette tip, transfer 800μL of supernatant from the culture tube into each corresponding cluster tube in the cluster rack. Be careful not to disturb the cell pellets.
- Cap the cluster tubes and store in the –80°C freezer.
T regulatory cell surface staining
(non-sterile technique)
- Add 1mL of staining buffer to each tube and vortex.
- Centrifuge cells at 300 g for 10 minutes at 4°C. Decant supernatant.
- Prepare 145 μL mAb cocktail in eppendorf tube:
- CD25-PCy5 (30 μL)
- CD4-PC7 (15 μL)
- CD3-APC7 (15 μL)
- 85 μL staining buffer
- Vortex. Store in fridge (light-sensitive) until ready for use]
- 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 Lysing Solution 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
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- 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.