Cruzfluor 555 cfl 555 conjugated anti rabbit igg

After placing sterile coverslips in a 6-well plate, HCE cells (3 × 10⁵ cells/well) were incubated overnight in KGM Bulletkit medium at 37 °C with CO₂. The next day, HCE cells were co-cultured with A. castellanii trophozoites and cysts (5 × 10⁵ cells/well) at 37 °C for 4 h. F. solani, P. aeruginosa, and S. aureus were cultured until the early exponential phase (OD_600nm = 0.8) and incubated with HCE cells and A. castellanii for 1 h. After discarding the culture media, cells were washed three times with ice-cold autoclaved PBS and then fixed with cold methanol for 5 min at RT. After repeated washing with PBS for 5 min, cells were incubated in a blocking buffer solution containing 1% BSA and 22.5 mg/mL glycine in PBS with 0.05% Tween 20 (PBST) for 30 min at RT. Afterward, cells were incubated overnight at 4 °C with CM antibody (1:200 dilution in blocking buffer). The next day, cells were washed with PBS and probed with CruzFluorTM 555 (CFL-555)-conjugated anti-rabbit IgG (1:400 dilution in blocking buffer) (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h at RT. After washing with PBS, cells were stained with VECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Newark, CA, USA) and observed under a fluorescence microscope (Leica, Wetzlar, Germany).

HCE cells (3 × 10⁵ cells/well) were cultured on sterile cover glass in a 6-well plate with KGM at 37 °C with 5% CO₂ for 24 h. The next day, A. castellanii trophozoites (5 × 10⁵ cells/well) or cysts (5 × 10⁵ cells/well) were added and co-cultured for 4 h. F. solani, P. aeruginosa, and S. aureus were cultured in the early exponential phase (OD_600nm = 0.8) and co-cultured with HCE cells and A. castellanii for 1 h. Cells were washed with ice-cold phosphate-buffered saline (PBS) and then fixed with 100% methanol (chilled at −20 °C) for 5 min. The cells were washed three times with PBS and permeabilized with PBST (0.2% Triton X-100 in PBS) for 5 min at room temperature (RT). Cells were blocked for 30 min at RT in blocking buffer (1% bovine serum albumin and 22.52 mg/mL glycine in PBST). Cells were incubated overnight at 4 °C with the ACAP or PBP antibodies in blocking buffer (1:200) and probed with CruzFluorTM 555 (CFL 555)-conjugated anti-rabbit IgG (1:400) (Santa Cruz, Dallas, TX, USA) for 2 h at RT. After washing three times with PBS, cells were stained with VECTASHIELD mounting medium (Abcam, Burlingame, CA, USA) and observed under a fluorescence microscope (Leica DMi8, Wetzlar, Germany).