A. Stimulation of the Cells
Various methods have been reported for the polarization and stimulation of T helper cell subsets to produce IL-17 in vitro. Short term activation with phorbol myristate acetate (PMA; protein kinase C activator) plus ionomycin (Ca2+ ionophore) has been useful for quickly inducing and characterizing polyclonal cytokine-producing cells. However, ex vivo stimulation of freshly explanted lymphoid cells from mice with PMA and ionomycin in culture for several hours typically results in the detection of only a small percentage of IL-17A-producing cells. For this kit, we recommend a 5 day polarization of enriched CD4+ T-cells cultured in the presence of TGFβ followed by a 4 hour restimulation with PMA + ionomycin in the presence of BD GolgiStop™ Protein Transport Inhibitor.
Note: Kinetic studies need to be performed to determine the optimal incubation time for each experimental system.
Procedure for generation of mouse IL-17A producing cells
1. Prepare a single cell suspension of splenocytes from a BALB/c mouse.
2. Lyse the red blood cells (1× BD PharmLyse™ Lysing Buffer, Cat. No. 555899).
3. Isolate CD4+ T cells by either a negative or positive cell selection method.
4. Culture cells for four days in the presence of plate-bound anti-CD3 antibody (Cat. No. 553057; 10 μg/ml; coat tissue culture plate wells
overnight at 4°C; wash wells three times with Dulbecco's PBS), soluble anti-CD28 antibody (Cat. No. 553294; 2 µg/ml final concentration),
and recombinant mouse IL-1β (Cat. No. 554577; 50 ng/ml), mouse IL-6 (Cat. No. 554582; 25 ng/ml), and TGFβ(Cat. No. 356039; 5 ng/ml)
proteins in complete RPMI-1640 tissue culture medium. Add fresh media at day 3 or 4 if necessary.
5. On day 5 harvest cells and wash once with complete RPMI-1640 tissue culture medium.
6. Restimulate cells with 50 ng/ml of PMA (Sigma Cat. No. P-8139) and 1 µg/ml of ionomycin (Sigma Cat. No. I-0634) in the presence of
Monensin (BD Golgistop™ Protein Transport Inhibitor (provided) Cat. No. 554724) in complete RPMI-1640. Incubate cells for four to five
7. Harvest cells.
8. Wash cells once in BD Pharmingen™ Stain Buffer (FBS)* (Cat. No. 554656).
9. Fix cells according to the Foxp3 staining procedure described below.
Optional (Cells may be frozen and stored for later use after fixation)
1. Fix cells with 1× BD Pharmingen™ Mouse Foxp3 Fixation Buffer (provided, Cat. No. 560409) at a concentration of 10 million cells/ml for
30 minutes on ice.
2. Wash cells once in BD Pharmingen™ Stain Buffer (FBS)* (5ml wash buffer per 10 million cells).
3. Count cells and suspend the cells at 10 million cells/ml in 10% DMSO/90% FCS.
4. Freeze cells at -80°C in a freezer vial.
B. Staining of the Cells
Collect cells from stimulatory cell cultures by centrifugation (300xg) and suspend them in BD Pharmingen Stain Buffer (FBS)*. Count the cells and adjust their concentration to 10 million cells/ml in BD Pharmingen Stain Buffer (FBS)*.
For frozen cells, remove DMSO by washing thawed cells with 5 ml/frozen vial with BD Pharmingen Stain Buffer (FBS)*, and centrifuge 300xg for 5 minutes at RT. Suspend pellet at 10 million cells/ml in BD Pharmingen Stain Buffer (FBS)* and aliquot 1 million cells per well of a 96 well plate. Centrifuge, remove buffer and continue procedure at step 7.
Note: This procedure is for staining cells in 96-well U bottom plates. If staining cells in tubes is preferred, then adjust wash volumes to 1 ml.
1. Remove clumps of cells and/or debris by passing the cell suspension through a BD Falcon™ 70-μm nylon cell strainer (Cat. No. 352235).
2. Dilute the cells to 10 million cells/ml.
3. Aliquot 1 million cells per well, centrifuge at 300xg for 5 minutes, and remove buffer.
4. To fix the cells, gently suspend the cell pellet in the residual volume of staining buffer and then add 200 µl of freshly prepared cold 1× BD
Pharmingen™ Mouse Foxp3 Fixation Buffer (provided, Cat. No. 560409). Mix well. Incubate for 30 minutes at 4°C in the dark.
5. Centrifuge 300xg for 5 minutes, and remove fixative.
6. To wash cells, suspend each pellet in 200 µl of freshly prepared pre-warmed (37°C) 1× BD Pharmingen™ Mouse Foxp3 Permeabilization
Buffer (provided, Cat. No. 560409), and centrifuge 300xg for 5 minutes. Remove permeabilization buffer.
7. To permeabilize the cells, gently suspend the cell pellet in another 200 µl of freshly prepared pre-warmed (37°C) 1× BD Pharmingen™
Mouse Foxp3 Permeabilization Buffer. Incubate for 30 minutes at 37°C in the dark.
8. Centrifuge the cells at 300xg for 5 minutes, and remove buffer.
9. To wash cells, add 200 µl of BD Pharmingen™ Stain Buffer (FBS) to each tube, centrifuge 300xg for 5 minutes. Remove buffer. Repeat.
10. Add 20 µl/test of the mouse Th17/Treg phenotyping cocktail or appropriate negative staining control. Incubate at RT for 30 minutes in the
dark. Cells should be protected from light throughout the staining and storage.
11. Repeat wash step 9 above two times.
12. Suspend cell pellet in 200 µl stain buffer and proceed with flow cytometric analysis.
C. Flow Cytometric Analysis
Set PMT voltage and compensation using unstained cells and appropriate cell surface markers
Note: It has been reported that CD4 expression on T cells is decreased after cell activation.
* BD Pharmingen Stain Buffer (FBS) (Cat No. 554656) is recommended for initial surface staining and all wash steps and covering tubes during incubation steps with caps or parafilm.
Warnings & Precautions
Danger: BD GolgiStop™ Protein Transport Inhibitor (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: Mouse Foxp3 Fixation Concentrate (20x) (component 51-9006124) 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.