inverted fluorescence phase contrast microscopy  (Danaher Inc)

Journal: Frontiers in Immunology

Article Title: M protein ectodomain-specific immunity restrains SARS-CoV-2 variants replication

doi: 10.3389/fimmu.2024.1450114

Figure Lengend Snippet: ADCC reporter assay. (A) Immunostaining of SARS-CoV-2-infected A549-TMPRSS2-ACE2 cells, using S2M2-30-specific hyperimmune serum. The uninfected cell culture was used as the negative control in immunostaining analysis. Phalloidin-iFluor dye (red) and DAPI (blue) were used for staining F-actin and nucleus in cells, respectively. A fluorescently labeled secondary antibody, Alexa Fluor 488 Goat Anti-Mouse IgG H&L (green), was utilized for signal amplification in the detection of the primary anti-S2M2-30 mouse serum antibodies. (B) Confocal fluorescence microscopy analysis of the transfected HEK293T cells overexpressing the recombinant M-GFP fusion protein (green) composed of the M protein and GFP. DIL dye (red) and DAPI (blue) were used for staining cellular membranes and nuclei in cells, respectively. (C) Confocal fluorescence microscopy analysis of the S2M2-30-specific serum-immunostained HEK293T cells overexpressing the M-GFP fusion protein (green). Fluorescently labeled secondary antibody Cy3 Goat Anti-Mouse IgG H&L (red) was utilized for signal amplification of primary mouse antibody detection (anti-S2M2-30 or anti-KLH serum). (D) ADCC surrogate assay with mAbs and transiently transfected HEK293T target cells overexpressing M protein (n = 2). An influenza hemagglutinin-specific mAb CR6261 was used as negative control. RLU, Relative light units. Data are presented as mean ± SEM. Multiple t -test were performed to statistically analyze the significance between the Preimmune group and S2M2-30-KLH group, as well as between the mAb CR6261 group and mAb 3M1C11 group. The *** represents P values less than 0.001.

Article Snippet: The lipid complex was wise-dripped into each well and incubated at 37°C, 5% CO 2 for 6 h. Then the cells were cultured in DMEM medium supplemented with 10% FBS at 37°C, 5% CO 2 for an additional 24 h. The successful expression of the M-GFP fusion protein was confirmed by observing green fluorescence using an inverted phase contrast fluorescence microscopy (Nikon, Cat. #TS2R-FL, Japan).

Techniques: Reporter Assay, Immunostaining, Infection, Cell Culture, Negative Control, Staining, Labeling, Amplification, Fluorescence, Microscopy, Transfection, Recombinant, Surrogate Assay