transwell cell culture inserts  (Sarstedt)

Journal: Infection and Immunity

Article Title: Differential sensitivity of leukocyte populations to Staphylococcus aureus biofilm

doi: 10.1128/iai.00654-25

Figure Lengend Snippet: Direct contact with S. aureus biofilm is critical to induce leukocyte cytotoxicity. Primary Mφs, G-MDSCs, and PMNs were co-cultured with S. aureus biofilm either directly or separated by Transwell inserts for the indicated intervals. Leukocytes were stained with anti-CD45, Zombie NIR (viability), Apotracker Green (phosphatidylserine), and MitoSOX Red (mtROS) to quantify ( A ) leukocyte viability, ( B ) early apoptosis, ( C ) late apoptosis/necrosis, and mtROS levels in ( D ) live and ( E ) early apoptotic cells. Unstimulated leukocytes were incubated in medium for 30 min or 2 h, as indicated ( n = 9 from three independent experiments; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Dunnett’s multiple correction between cell types).

Article Snippet: On day 4 of biofilm growth, ~45% of the medium was removed and leukocytes were added at a density of 2.5 × 10 5 cells/well in fresh medium and incubated for 15 min, 30 min, 2 h, and 6 h. For Transwell co-cultures, Transwell inserts (0.4 μm; CELLTREAT #230635) were placed in 24-well plates above the biofilm on day 4 of growth, whereupon 7.5 × 10 5 leukocytes were added to the upper chamber for 30 min or 2 h. Leukocytes were stained and acquired as described above for planktonic co-culture studies, and unstimulated cells were included for the duration of the co-culture period.

Techniques: Cell Culture, Staining, Incubation