A. Stimulation of the Cells
Various in vitro methods have been reported for polarization or stimulation of T helper cells subsets of which PMA (Phorbol ester) plus Ionomycin (Calcium Ionophore) has been particularly useful for quickly inducing and characterizing polyclonal cytokine-producing cells. For this kit we recommend the stimulation of normal PBMCs at a concentration of 1-10 million cells per ml in media for 5 hours with PMA/Ionomycin (at 50 ng/ml and 1 μg/ml respectively) in the presence of BD GolgiStop™ Protein Transport Inhibitor (provided in the kit or Cat #554724). Add 4 μl of BD GolgiStop™ for every 6 ml of cell culture and mix thoroughly. It is recommended that BD GolgiStop™ not be kept in cell culture for longer than 12 hours.
Note: Kinetic studies need to be performed to determine the optimal incubation time for each experimental system. Depending on the donor, frequencies of cytokine producing cells derived from activation of PBMC can vary widely for a specific cytokine. In particular, the number of IL-17 producing cells can be very low or even negligible on PMA/Ionomycin stimulated PBMC. In these cases, Th17 polarization cultures should be considered. For specifics on polarization of Th17 cells please refer to the references.
B. Staining of the Cells
1. Harvest of the Cells
Collect cells from in vitro stimulatory cultures treated with a protein transport inhibitor. Spin down cells at 250 x g for 10 minutes at room temperature (RT) and wash two times with stain buffer (FBS) (Cat# 554656). Count cells and transfer approximately 1X10^6 cells to each flow test tube (Cat# 352008) for immunofluorescent staining. Cells should be protected from light throughout the staining procedure and storage.
2. Fixing the Cells
a. Spin down cells at 250 x g for 10 minutes at RT and thoroughly suspend cells with 1ml of cold BD Cytofix™ Fixation Buffer (provided in the kit or Cat# 554655) and incubate for 10-20 minutes at RT.
Note: Cell aggregation can be avoided by vortexing prior to the addition of the fixative.
b. Spin down cells at 250 x g for 10 minutes at RT.
Note: After fixation, the cell pellet after centrifugation is loose and care should be taken when aspirating the wash buffer from the tubes. Do not aspirate ALL of the buffer but leave 50-150 µl of solution in the tubes to avoid cell loss for all subsequent wash steps below.
c. Wash cells twice at RT in stain buffer (FBS) and spin down the cells at 250 x g for 10 minutes at RT.
Note: Cells can be stored in stain buffer at 4°C for up to 72 hours or in 90% FCS/10% DMSO at -80°C for up to six months (for samples that need to be stored longer than six months, we recommend performing stability studies) .
3. Permeabilizing the Fixed Cells
a. For cells kept at 4°C, spin down cells at 250 x g for 10 minutes at RT and remove stain buffer.
b. For cells stored at -80°C thaw and wash twice with stain buffer (FBS) to remove DMSO.
c. Dilute 10x BD Perm/Wash™ buffer (provided in the kit or Cat# 554723) in distilled water to make a 1x solution prior to use.
d. Suspend cells in 1ml of 1x BD Perm/Wash™ buffer and incubate at RT for 15 minutes.
e. Spin down cells at 250 x g for 10 minutes at RT and remove supernatant.
4. Staining with the Cocktail
a. Thoroughly suspend fixed/permeabilized cells in each tube in 50 μl of BD Perm/Wash™ buffer and add 20 ul/tube of cocktail or appropriate negative control. Incubate at RT for 30 minutes in the dark. Cells should be protected from light throughout the staining procedure and storage.
b. Optional: Staining of Additional Cell Surface Antigens
Note: If instrument allows, optional multicolor staining of different cell surface antigens can be done at this time. Example: PE-Cy™7 CD3 (Clone SK7, Cat# 557851), V450 CD8 (Clone RPA-T8, Cat# 560347) and V450 CD45RA (Clone HI100, Cat# 560362) etc. For cell surface staining after fixation/permeabilization, suitable antibody clones that recognize denatured epitopes need to be identified.
Note: For antibodies that do not recognize fixed/denatured cell surface markers, it is recommended that staining be done on live cells PRIOR to fixation/permeabilization.
c. Wash cells twice with 1ml of 1× BD Perm/Wash™ buffer at RT and suspend in Stain Buffer (FBS) prior to flow-cytometric analysis.
C. Flow Cytometric Analysis
Set PMT voltage and compensation using unstained cells and appropriate cell surface markers or use BD™ Compensation beads (Cat # 552844; 560499) as per the recommended protocol.
Note: It has been reported that CD4 expression on T cells is decreased after cell activation.
Note: Acquire at least 20,000 to 30,000 CD4 positive lymphocytes. Depending on the donor, frequencies of cytokine producing cells derived from activation of human PBMCs can vary widely for a particular cytokine. In order to make statistically significant frequency measurements, sufficiently large sample sizes should be acquired during flow cytometric analysis. Bivariate dot plots or probability contour plots can be generated upon data reanalysis to display the frequencies of and patterns by which individual cells co-express certain levels of cell surface antigen and intracellular cytokine proteins.
Danger: BD GolgiStop™ Protein Transport Inhibitor, containing monensin (component 51-2092KZ) contains 99.61% ethanol (w/w) and 0.26% monensin, mononatriumsalz (w/w).
Highly flammable liquid and vapor.
Causes serious eye irritation.
Keep away from heat/sparks/open flames/hot surfaces. No smoking.
Wear protective gloves / eye protection.
Wear protective clothing.
IF ON SKIN (or hair): Remove / Take off immediately all contaminated clothing. Rinse skin with water / shower.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
Dispose of contents / container in accordance with local / regional / national / international regulations.
Danger: BD Cytofix™ Fixation Buffer (component 51-9006613) contains 4.2% formaldehyde (w/w).
Harmful if inhaled.
Causes skin irritation.
Causes serious eye damage.
May cause an allergic skin reaction.
Suspected of causing genetic defects.
May cause cancer. Route of exposure: Inhalative.
May cause respiratory irritation.
Wear protective clothing / eye protection.
Wear protective gloves.
Do not breathe mist/vapours/spray.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.
If skin irritation or rash occurs: Get medical advice/attention.