Enspire series multilabel plate reader

Sf21 cells were infected with S-Bac or WT-Bac using an M.O.I. of 1 and incubated at 26 °C for 48 h. Insect cells, i.e., S-Bac-infected cells or WT-infected cells, were washed with PBS and seeded in the well of a 96-well microplate (1 × 10⁴/well) for 1 h at room temperature. The cells were fixed with 4% paraformaldehyde for 10 min, washed three times with PBST (PBS with 0.1% Tween 20), then blocked with 3% BSA for 1 h. Cells were incubated with diluted antisera for 2 h at 26 °C. After incubation, cells were washed three times with PBST and incubated with Peroxidase AffiniPure goat anti-swine IgG (1:5000 dilution, Jackson ImmunoResearch) as secondary antibody for signal detection following reaction with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate for 1 h. The plates were read at 450 nm using an EnSpire Series Multilabel Plate Reader (PerkinElmer). Mean optical density (OD) values were used to quantify ELISA data.

Hi5 cells (1 × 10⁴) were seeded onto a 96-well plate (Falcon) and infected with recombinant baculovirus expressing Gp.Mur antigen using MOI = 1. At 3 days post infection (dpi), the cells were fixed with 4% paraformaldehyde and blocked with 3% bovine serum albumin (BSA) in DPBS for 1 h. The cells were then incubated for 2 h with mouse anti-His antibody (1:5000, GeneTex GTX628914) at 25 °C. After three washes with DPBS with 0.1% Tween^® 20 (Sigma-Aldrich, CO, USA) (DPBST), HRP-conjugated goat anti-mouse IgG secondary antibody was added (1:5000) and incubated for 1 h. The enzymatic reaction with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was stopped by adding 2N H₂SO₄ after 5 min of color development. The optical density (OD) of the Gp.Mur antigen expressed in insect cells was compared to that of WT-td-infected cells and uninfected cells (CO) to quantify surface expression of Gp.Mur antigen (EnSpire Series Multilabel Plate Reader, PerkinElmer).

SNU-1, the GC cell line, was purchased from the Bioresource Collection and Research Center and maintained in Roswell Park Memorial Institute Medium 1640 (Gibco, Brooklyn, NY, USA) containing 10% fetal bovine serum at 37 °C in 5% CO₂. The 2,3-bis (2-methoxy 4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanlide inner (XTT) assay was used to determine the viability of SNU-1 cells. The cells were cultured in 96-well culture plates (5 × 10³/well) overnight at 37 °C and then subjected to varying concentrations of escitalopram oxalate and 5-FU administered alone or in combination for another 24 and 48 h. After the incubation process was completed, the medium was removed, and fresh culture medium was added. Subsequently, a total of 50 µl XTT was added to each well of the 96-well culture plates and then incubated for another 4 h (Biological industries, Haemek, Israel). Finally, the absorbance was detected at a wavelength of 630–690 nm by using a microplate reader (EnSpire Series Multilabel Plate Readers, PerkinElmer Inc., MA, USA).