Live dead fixable violet dead cell stain kit

Chlamydial-specific T cell expansion and cytokine production from PBMC was done as described (14 (link)). Briefly, CFSE (carboxy fluorescein succimidyl ester, Molecular Probes) labeled PBMCs were incubated at 2 x10⁶ cell/ml in deep well tissue culture plates (96 well, Eppendorf) in 1 ml of AIM V medium (Life Technologies) only or pulsed with C. trachomatis A2497P⁺ elementary bodies (EB) (MOI= 20) or chlamydial soluble antigen (SA) (100 μg/ml) and incubated for five days at 37°C and 5% CO₂. Cells were then re-stimulated for six additional hours with medium only or chlamydial antigens in the presence of mAb co-stimulatory signals. The mAb used were 0.5 μg/ml of anti-CD28 (clone CD28.2, Nonhuman Primate Reagent Resource, University of Massachusetts Medical School, Boston, MA), Mab CD49d (clone 9F10, BD Biosciences) and 1X brefeldin A solution (BD Biosciences). The cells were washed and analyzed by flow cytometry. A Live/Dead fixable violet dead cell stain kit (Molecular Probes) was used to discriminate live and dead cells. Dilution of CFSE stain was used to monitor proliferation of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. The percent of cells proliferating was normalized against their respective negative controls which were cells incubated with sucrose-phosphate glutamate buffer (SPG) or SA prepared from mock-infected BGMK monolayers.

Sucrose, glucose, HEPES (4–2(2-hydroxymethyl)-1-piparazineethane-sulfonic acid), sodium chloride, and hydrochloric acid (HCl) were purchased from Fisher Scientific. Ammonium sulfate, sodium phosphate, potassium phosphate, potassium chloride, sodium bicarbonate, lectin protein from Canavalia ensiformis, ATTO-488 NHS-ester, TCEP (tris(2-carboxyethyl) phosphine hydrochloride), and doxorubicin were purchased from Sigma-Aldrich. Fetal bovine serum (FBS), trypsin, penicillin, streptomycin, L-glutamine, PBS (phosphate buffered saline), and DMEM (Dulbecco’s modified Eagle medium) were purchased from GE Healthcare. Texas Red-DHPE (Texas Red-1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine) was purchased from Thermofisher. Trypan blue was purchased from Life Technologies. DPPC (1,2 – dipalmitoyl-sn-glycero-3-phosphocholine), DOPC (1,2 dioleoyl-sn-glycero-3-phosphocholine), cholesterol (from ovine wool), DOTAP (1,2 dioleoyl--3-trimethylammonium-propane), and PEG2000-DPPE (1,2 dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000]) were all purchased from Avanti Polar Lipids (Alabaster, AL). LIVE/DEAD fixable violet dead cell stain kit was purchased from Thermo Fisher Scientific. All reagents were used without further purification.