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| ==Overview== | | ==Overview== |
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| This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.
| | Replace this sentence with a brief description of the protocol and its goal. |
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| ==Materials== | | ==Materials== |
| *List reagents, supplies and equipment necessary to perform the protocol here.
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| {| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse; width:710px" <!--
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| | For a 50 μL PCR reaction: |
| | align="center" style="background:#f0f0f0;"|'''Isolation of Mononuclear Cells'''
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| | align="center" style="background:#f0f0f0;"|'''PBMC Antigen Stimulation Assay'''
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| | align="center" style="background:#f0f0f0;"|'''Collection of Supernatant'''
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| | align="center" style="background:#f0f0f0;"|'''T-Regulatory Cells Surface Staining'''
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| |--
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| |*On average, 18mL of whole heparinized blood/patient will be given.
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| |*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)
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| |*Sterile 5mL polystyrene round-bottom tubes.
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| |*Phosphate buffered saline (PBS)
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| |--
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| |--
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| |*Ficoll Paque Plus [Endotoxin tested], room temperature.
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| |*CFSE (5μM aliquots).
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| |*Staining buffer (PBS + 5g BSA/L + 2mM EDTA)
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| |*Staining buffer (PBS + 5g BSA/L + 2mM EDTA)
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| |--
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| |--
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| |*Sterile phosphate buffered saline (PBS), room temperature
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| |*AIM-V Media [add 500μL Amphotericin (Fungizone), 5mL PS (PCN streptomycin), and 5mL Glutamine].
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| |*Cluster tubes or Cryovial tubes and racks.
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| |*Cell separation buffer (10X stock; prepare 1X using distilled water)
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| |--
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| |--
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| |*0.2% and 0.4% Trypan solution.
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| |*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°].
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| |*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]
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| |--
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| |*Sterile conical tubes (15mL, 50mL)
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| |*CD3, CD28 expander beads
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| |*1X Facs Lysis Buffer
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| |--
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| |*Sterile, graduated transfer pipettes.
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| |*Mus M1 (50µl) aliquot tube.
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| |*A solution of 20% DMSO + PBS.
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| |--
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| |*Hemocytometer or disposable slides for the automated counter or manual counter
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| |*Tetanus Toxoid (stock 2 mg/mL).
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| |*Fix/Perm Concentrate
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| |*Accuspin tubes
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| |*24 well tissue culture plates.
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| |*Fix/Perm Diluent
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| |*Sterile 5mL polypropylene round-bottom tubes
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| |*FoxP3-Alexa 647 Antibody.
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| |*15mL conical tubes.
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| |*5mL polystyrene tubes
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| |--
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| =='''Procedure'''==
| | * 35 μL H<sub>2</sub>O |
| | * 5 μL 10X PCR buffer |
| | * 5 μL 2mM dNTPs (each) |
| | * 1.5 μL 50mM MgCl<sub>2</sub> |
| | * 1 μL 50μM sense primer |
| | * 1 μL 50μM antisense primer |
| | * 1 μL 5nM DNA template |
| | * 0.5 μL TAQ DNA polyermerase |
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| ==Isolation of Mononuclear Cells== | | ==Procedure== |
| | | # In a PCR tube, mix the components on ice in the order they are listed above. |
| * This is a sterile procedure and all steps should be performed in a hood.
| | # Perform thermocycling program |
| | | ## 95 °C 5 min |
| # Turn on the hood. Bring Ficoll and PBS to room temperature in the hood. | | ## 95 °C 30 s |
| | | ## T<sub>H</sub> 30 s |
| # Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and record subject information, i.e. ID #, date collected, date received.
| | ## 72 °C 1 min for each 1 kb PCR product |
| | | ## Repeat steps 2-4 a total of 12-36 times (24 is standard). |
| # If performing Basophil Activation assay on this sample, set aside 3mL of whole blood.
| | ## 72 °C 5 min |
| | | ## 12 °C hold |
| # Dilute remaining blood at 1:1 with PBS in 50 mL conical tubes, adding PBS first followed by blood.
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| # Place 15 mL of Ficoll in a 50 mL conical tube. Overlay with up to 30mL of diluted blood, adding it very slowly to make sure that the blood doesn’t mix with the Ficoll layer.
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| # Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
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| # 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.
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| # Centrifuge at 500 g for 20 minutes at room temperature (maximum acceleration and deceleration).
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| # Aspirate and discard the supernatant. Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding 1 mL of PBS. Set aside a 10 µL aliquot of cells for counting as follows: place the 10 µL of cells into 90 µL of PBS in a small sterile eppendorf tube, and add 100 µL of Trypan Blue (0.2%) into the eppendorf tube. Mix well, and let it sit for 1 minute before counting.
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| # Add 19 mL of PBS to the cells in 990 µL to make a total volume of 19.99 mL and centrifuge at 300 g for 15 minutes at room temperature (maximum acceleration and deceleration).
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| # 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.
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| # 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.
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| # 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 10^6/ml in 15 ml PP conical tube.
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| =='''PBMC Antigen Stimulation Assay'''==
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| ==*This is a sterile procedure and all steps should be performed in a hood.==
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| # Remove stimulants from the freezer and thaw.
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| # 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). | |
| ## Musm1 (allergen): @ 200μg/mL purified Musm1 protein in Aim-V. | |
| ## AIM-V + IL-2 (negative control): AIM-V + IL-2 medium alone. | |
| ## Beads (positive control): 1μg/mL anti-Cd3/Cd28 beads. | |
| ## Tetanus: 200μg/mL tetanus in AIM-V. | |
| # Add an equal volume (1:1 dilution) of freshly prepared 10μM CFSE (in PBS) to the tube of cells. To make 1.5mL of PBS + CFSE (2x solutin): add 3μL of stock CFSE (5mM) into 1.5mL of PBS.
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| # Incubate in 37deg;C water bath for 10 minutes.
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| # After incubation, wash the CFSE stained cells in 10mL of AIM-V @ 300g for 10 minutes. Aspirate supernatant after centrifugation.
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| # Resuspend CFSE stained cells in AIM-V medium @ 4 million cells/mL. For plating, each well should contain 2 milliion cells/mL.
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| * Begin the stimulation process by preparing AIM-V medium + a 2X solution of IL-2 by adding 2μL of IL-2 per mL of medium in a 15mL conical tube. For 5 stimulant conditions, you will need atleast 2.5mL of AIM-V + IL-2. Vortex gently.
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| # Prepare solutions for each stimulant condition in 5 mL PP tubes as follows:
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| ## AIM-V+IL-2 medium alone: place 500μL of AIM-V+IL-2 in the appropriately labeled tube and add 500μL of cells in AIM-V+IL-2. Pipette up and down and plate.
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| ## 1μg/mL anti-CD3, anti-CD28 beads in AIM-V+IL-2: place 500μL of AIM-V in the appropriately labeled tube and add 2.5μL of CD3, CD28 expander beads. Then add 500μL of cells in AIM-V+IL-2. Pipette up and down and plate.
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| ## 200μg/mL MusM1 in AIM-V+IL-2: place 450μL of AIM-V+IL-2 in the appropriately labeled tube and add 50μL of allergen (MusM1). Then add 500μL of cells in AIM-V+IL-2 + allergen. Pipette up and down, and plate.
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| ## 200μg/mL tetanus in AIM-V+IL-2: place 475μL of AIM-V+IL-2 in the appropriately labeled tube and add 25μL of tetanus. Then add 500μL of cells in AIM-V+IL-2. Pipette up and down and plate.
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| ## Place the tissue culture plate in the 37°C CO2 incubator for 7 days.
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| =='''COLLECTION OF SUPERNATANTS'''==
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| # Cell culture supernatants will be collected for cytokine measurement for 2 or 7 days cultures.
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| # At day 7, collect cells carefully by pipetting up and down to resuspend cells in the well, and
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| transfer the total volume in each well into separate 5ml polystyrene tubes. Rinse each well
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| with 200μl of staining buffer and add to corresponding PS tubes. Tap gently to mix.
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| # Centrifuge tubes at 300g for 5 minutes at 25°C temperature.
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| # Obtain a cluster tube rack for the storage of supernatants.
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| # Label cluster tubes as follows:
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| ## Specimen ID
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| ## Date
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| ## Supernatants - 7 day
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| ## Stimulant condition 1-4
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| ### Stimulant 1 = MusM1
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| ### Stimulant 2 = AIM-V | |
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| ### Stimulant 3 = Beads
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| ### Stimulant 4 = Tetanus Toxoid
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| ## 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.
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| ## Cap the cluster tubes and store in the –80oC freezer (by the freight elevators).
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| =='''T REGULATORY CELL SURFACE STAINING'''==
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| # After the collection of supernatants according to the Collection of Supernatants Protocol. Resuspend cells in 80μL of cell separation buffer.
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| # Add 1mL of staining buffer to each tube and vortex. | |
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| # Wash cells at 300 g for 5 minutes at 4°C. Decant supernatant. | |
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| # Add 1mL of staining buffer to each tube and vortex.
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| # Wash cells at 300 g for 10 minutes at 4°C. Decant supernatant. | |
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| # Prepare cocktail preparation according to the T REGULATORY CELL SURFACE STAINING; Materials. Add 50μL of cocktail to each tube. | |
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| # Incubate @ 4°C for 20-30 minutes.
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| # Add 3mL of staining buffer to each tube and vortex.
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| # Wash at 300 g for 10 minutes at 4°C. Decant supernatant.
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| * At this point in the experiment, one can fix/freeze the cells or continue with Intracellular cytokine staining.
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| ## 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. After the incubation, pipette 500μl of the 20% DMSO + PBS solution into each tube.
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| # Transfer the 1ml of cells + 1X Facs Lysis Buffer + 20% DMSO/PBS solution into an appropriately labeled cluster tubes, and freeze at -80°C until when you are ready to conduct Intracellular cytokine staining..
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| # To Intracellular Cytokine stain the cell with Fox P3, prepare a Fix/Perm working solution as follow: dilute the Fix/Perm Concentrate (1 part) into the Fix/Perm Diluent (3 parts) to the desired volume of working solution (1mL per tube).
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| # Resuspend cell pellet with pulse vortex and add 1mL of Fix/Perm buffer to each sample tube.
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| Pulse vortex again.
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| # Incubate at 4°C for 30-60 minutes in the dark (preferably for 60 minutes if possible).
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| Wash for 10 minutes at 800 g x 2 (4°C) with 2mL of 1X Perm Buffer (made from 10X solution using dH2O). Decant supernatant.
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| # Add 20μL of Foxp3 antibody into 100μL of 1X Perm Buffer. Incubate for at least 30 minutes (45
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| minutes if possible) at 4°C.
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| # Wash at 800 g for 10 minutes x 2 with 2mL 1X Perm Buffer. Decant supernatant. Vortex and acquire using the Flow machine at 17-40.
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| ==Notes== | | ==Notes== |
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| ==References== | | ==References== |
| '''Relevant papers and books''' | | '''Relevant papers and books''' |
| <!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. --> | | <!-- If this protocol has papers or books associated with it, list those references here.--> |
| | <!-- See the [[OpenWetWare:Biblio]] page for more information, but this format doesn't work currently. |
| <biblio> | | <biblio> |
| #Goldbeter-PNAS-1981 pmid=6947258 | | #Goldbeter-PNAS-1981 pmid=6947258 |
| #Jacob-JMB-1961 pmid=13718526 | | #Jacob-JMB-1961 pmid=13718526 |
| #Ptashne-Genetic-Switch isbn=0879697164 | | #Ptashne-Genetic-Switch isbn=0879697164 |
| </biblio> | | </biblio>--> |
| | <!-- Try the [[Template:FormatRef|FormatRef template]]--> |
| | #{{FormatRef|Goldbeter, A and Koshland, DE|1981| |Proc Natl Acad Sci U S A 78(11)|6840-4| }} PMID 6947258 |
| | #{{FormatRef|Jacob, F and Monod, J J|1961| |Mol Biol 3(3)|318-56| }} PMID 13718526 |
| | #{{FormatRef|Ptashne, M|2004|Genetic Switch: Phage Lambda Revisited| | |Cold Spring Harbor Laboratory Press}} ISBN 0879697164 |
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| ==Contact== | | ==Contact== |