Shreffler:JAX Tcell studies: Difference between revisions
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(New page: ==Overview== This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study. ==Materials== *List reagents, supplies and equipment necessary to perform the protocol...) |
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==Materials== | ==Materials== | ||
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'''Isolation of Mononuclear Cells''' | |||
'''PBMC Antigen Stimulation Assay''' | |||
'''Collection of Supernatant''' | |||
'''T-Regulatory Cells Surface Staining''' | |||
*On average, 18mL of whole heparinized blood/patient will be given. | |||
*8 x 10^6 (8 million) cells for 7 day culture (for measurement of frequency of PBMC-derived, Mus M1-specific CD4+ CD25+ FoxP3+ T cells) | |||
*Sterile 5mL polystyrene round-bottom tubes. | |||
*Phosphate buffered saline (PBS) | |||
*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 | |||
*AIM-V Media [add 500μL Amphotericin (Fungizone), 5mL PS (PCN streptomycin), and 5mL Glutamine]. | |||
*Cluster tubes or Cryovial tubes and racks. | |||
*Cell separation buffer (10X stock; prepare 1X using distilled water) | |||
*0.2% and 0.4% Trypan solution. | |||
*IL-2 (approximately 20 units/μL; 1 μg = 2.4x10^3 units) [To make a 10μg 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°]. | |||
*Cocktail preparation [need 50μL of cocktail/flow tube; volume of mAb needed per flow tube: CD25-PCy5 (10uL), CD4-PC7 (5uL), CD3-APC7 (5uL), Aqua live/ dead (2μL),CD127-PE (1uL); add staining buffer to “cocktail tube” to reach a total volume of 50μl per flow tube. Vortex. Store in fridge (light-sensitive) until ready for use] | |||
*Sterile conical tubes (15mL, 50mL) | |||
*CD3, CD28 expander beads | |||
*1X Facs Lysis Buffer | |||
*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 | |||
*Tetanus Toxoid (stock 2 mg/mL). | |||
*Fix/Perm Concentrate | |||
*Accuspin tubes | |||
*24 well tissue culture plates. | |||
*Fix/Perm Diluent | |||
|Sterile 5mL polypropylene round-bottom tubes | |Sterile 5mL polypropylene round-bottom tubes | ||
*FoxP3-Alexa 647 Antibody. | |||
*15mL conical tubes. | |||
*5mL polystyrene tubes | |||
=='''Procedure'''== | =='''Procedure'''== |
Revision as of 07:56, 4 November 2009
Overview
This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.
Materials
Isolation of Mononuclear CellsPBMC Antigen Stimulation AssayCollection of SupernatantT-Regulatory Cells Surface Staining- On average, 18mL of whole heparinized blood/patient will be given.
- 8 x 10^6 (8 million) cells for 7 day culture (for measurement of frequency of PBMC-derived, Mus M1-specific CD4+ CD25+ FoxP3+ T cells)
- Sterile 5mL polystyrene round-bottom tubes.
- Phosphate buffered saline (PBS)
- 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
- AIM-V Media [add 500μL Amphotericin (Fungizone), 5mL PS (PCN streptomycin), and 5mL Glutamine].
- Cluster tubes or Cryovial tubes and racks.
- Cell separation buffer (10X stock; prepare 1X using distilled water)
- 0.2% and 0.4% Trypan solution.
- IL-2 (approximately 20 units/μL; 1 μg = 2.4x10^3 units) [To make a 10μg 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°].
- Cocktail preparation [need 50μL of cocktail/flow tube; volume of mAb needed per flow tube: CD25-PCy5 (10uL), CD4-PC7 (5uL), CD3-APC7 (5uL), Aqua live/ dead (2μL),CD127-PE (1uL); add staining buffer to “cocktail tube” to reach a total volume of 50μl per flow tube. Vortex. Store in fridge (light-sensitive) until ready for use]
- Sterile conical tubes (15mL, 50mL)
- CD3, CD28 expander beads
- 1X Facs Lysis Buffer
- 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
- Tetanus Toxoid (stock 2 mg/mL).
- Fix/Perm Concentrate
- Accuspin tubes
- 24 well tissue culture plates.
- Fix/Perm Diluent
Sterile 5mL polypropylene round-bottom tubes
ProcedureIsolation of Mononuclear Cells
PBMC Antigen Stimulation Assay*This is a sterile procedure and all steps should be performed in a hood.
COLLECTION OF SUPERNATANTS
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.
T REGULATORY CELL SURFACE STAINING
NotesPlease feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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