Tsst 1

Phytohemagglutinin (PHA), lipopolysaccharide (LPS), Staphylococcal enterotoxin A (SEA), and toxic shock syndrome toxin-1 (TSST-1) (Sigma-Aldrich) were reconstituted in RPMI 1640 medium at 11 times the desired final concentration, filter sterilized, and stored in aliquots at −30 °C, as described previously [25 (link)]. Each mitogen/antigen was used at final in-use concentrations of 5 and 10 µg/mL.
Cells (200 µL) were seeded at 1 × 10⁵ cells/mL, 5 × 10⁵ cells/mL, and 1 × 10⁶ cells/mL into six replicate wells of a 96-well cell culture plate. Cells were stimulated to proliferate non-specifically for 72 h, with 20 µL of mitogen/antigen, while control unstimulated cells received 20 µL of complete medium. Cell proliferation was measured using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) colorimetric assay (Roche, Basel, Switzerland). Briefly, 20 µL of MTT labeling reagent (5 mg/mL in PBS) was added to each well and incubated for 6 h. The culture supernatant was then discarded, 10% SDS in 0.01 M HCl was added, and the amount of MTT formazan produced was measured at a wavelength of 570 nm. Cell proliferation was expressed as a stimulation index (SI), calculated as follows: $$StimulationIndex\left(SI\right)=\frac{O{D}_{570}ofstimulatedcells}{O{D}_{570}ofunstimulatedcells}$$

The S. aureus strain RN6390 was used to induce endophthalmitis [20 (link), 21 (link)]. The bacterial strain was maintained and grown in tryptic soy broth (Sigma Aldrich, St. Louise, USA) overnight at 37°C. The bacterial count was adjusted to 5000 cfu/ml in PBS. For the preparation of heat killed S. aureus (HKSA), 10⁵ cfu/ml of bacterial culture was boiled in a water bath for 10 min., followed by a viability assay using bacterial plating. Purified PGN, SPA, α-toxin, TSST1, and LTA from Staphylococcus aureus were purchased from Sigma Aldrich, USA. A dose response study was performed to select the suitable dose that worked for each bacterial virulence factor to elicit inflammation (Fig 1). Alpha-toxin was tested for hemolytic activity in 5% sheep blood agar before injection. All the virulence factors were dissolved in endotoxin-free water and checked for endotoxin levels prior to injection by using LIMULUS amoebocyte lysate assay (Genescript, NJ, USA). The endotoxin levels in LTA, PGN and TSST1 were <0.005 EU/μg while in α-toxin and SPA it was <0.05 EU/μg, of protein.