Cy3 conjugated goat anti mouse igg

The effectivity of mAb 2c to inhibit cell-to-cell transmission of the eye pathogenic strain HSV-1 KOS was analyzed by immunofluorescence as previously described [11 (link)]. Briefly, confluent monolayers of Vero cells, grown in 24-well tissue culture plates, were infected with 100 TCID₅₀ of HSV-1 KOS. After 4 h of adsorption at 37°C the viral inoculum was removed. Cells were incubated for 48 h in DMEM containing 2% FCS in the presence of 500 nM (75µg/ml) mAb 2c, pooled human sera (1:40 diluted in medium) derived from donors with high titers of anti-HSV-immunoglobulins (neutralizing titer 1:256 for total neutralization of 100 TCID₅₀ of HSV-1 KOS), or medium alone. The antibody concentrations in cell supernatants represent excess concentrations required for the complete neutralization of the secreted virus. Plaque formation was detected by immunofluorescence. HSV-1 infected cells were stained with a mouse anti-HSV-1/2-gD-antibody (Acris Antibodies, San Diego, CA, USA) and an Alexa Fluor 488 goat anti-mouse IgG specific secondary antibody (Invitrogen). Bound human or murine antibodies were stained with Cy3-conjugated goat anti-mouse IgG or goat anti-human IgG secondary antibodies (Invitrogen), respectively. Immunofluorescence images were acquired with a Zeiss Observer Z1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) at a 100-fold magnification.

To determine the influence of α-MG on the presence of DENV-2 NS5 and nuclear translocation of NF-κB p65, HepG2 cells were grown on glass coverslips for 24 h and infected with DENV-2 in the presence or absence of α-MG or RV as described above. After incubation at 37 °C in a 5% CO₂ atmosphere for 24 h, the treated cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS, and incubated with a mixture of mouse anti-DENV-NS5 antibody and rabbit anti-NF-κB p65 at RT for 1 h. After washing with PBS, the cells were incubated with a mixture of Cy3-conjugated goat anti-mouse IgG (Invitrogen), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen), and Hoechst 33342 for nuclear staining (Invitrogen) at RT for 1 h. Fluorescence images were visualized using a laser scanning confocal microscope (LSM 800; Zeiss Microscopy, Jena, Germany).
To investigate the influence of α-MG on the presence of DENV-2 NS5 and nuclear translocation of NF-κB p65 during the early phase of infection, HepG2 cells were infected with DENV-2 and treated with 20 μM of α-MG. The infected cells were harvested at 0, 1, 3, 6, and 24 h post-treatment. DENV-2 NS5 and NF-κB p65 were detected as described above.

To assess the impact of KG extract or EPMC on DENV infection, cells cultured on glass coverslips underwent a series of steps. First, they were fixed with a 4% paraformaldehyde solution in PBS, followed by permeabilization using 0.2% Triton X-100 in PBS. Subsequently, the cells were incubated with the 4G2 antibody. After thorough washing, a mixture containing Cy3-conjugated goat anti-mouse IgG (Invitrogen) was applied, along with Hoechst 33342 for nuclear staining (Invitrogen). The resulting fluorescence images were visualized using a laser scanning confocal microscope (LSM 800, Zeiss, Jena, Germany).
To investigate the influence of EPMC on the presence of DENV-2 NS5 and the nuclear translocation of NF-κB p65, the cells were subjected to a similar procedure. They were incubated with a mixture comprising mouse anti-DENV-NS5 and rabbit anti-NF-κB p65 antibodies, followed by a combination of Cy3-conjugated goat anti-mouse IgG, Alexa Fluor 488-conjugated donkey anti-rabbit IgG, and Hoechst 33342^{18 (link)}.

Rabbit anti-human phospho p65 NF-κB, PathScan^® Signaling Nodes Multi-Target Sandwich ELISA, PathScan^® signaling NF-κB ELISA, and PathScan^® Sandwich ELISA Lysis Buffer were obtained from Cell Signaling Technology (Pickering, ON, Canada). Cy3-conjugated goat anti-mouse IgG was purchased from Invitrogen Canada (Burlington, ON, Canada). Bordet-Gengou blood agar plates were from BD Biosciences (Mississauga, ON, Canada). Syto9 and Propidium Iodide were from ThermoFisher (Waltham, MA). Wild-type B. pertussis (Tohama I) was propagated at Sanofi Pasteur (Toronto, ON, Canada). Human lung carcinoma epithelial A549 cell line was obtained from ATCC (Old Town Manassas, VA). For the cytokine detection (Eotaxin, Eotaxin-3, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-8 (HA), IL-21, IL-22, IL-23, IL-27, IL-31, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, MIP-3α, TARC, TNF-α, TNF-β, VEGF-A, GM-CSF, and IFN-γ), V-PLEX Human Cytokine 36-Plex Kit (K15089D-2) was used for screening and only 20 selected cytokines were further tested on 20-plex cytokines (Meso Scale Discovery, Rockville, MD).

Vero cells were plated at a cell density of 6 × 10⁴ cells/well in a 24-well plate. Cells were infected with SARS-CoV-2 at an MOI of 1 in a Biosafety Laboratory Level 3 (BSL-3, Instituto Nacional de Enfermedades Respiratorias, Mexico city, Mexico). Cells were incubated for 24 h with 5% CO₂ at 37 °C, washed with 1X PBS, and fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Fixed cells were permeabilized with 0.1% Triton-X100 in PBS (PBS-Tr) for 30 min, blocked with 10% goat serum in PBS-Tr for 30 min, and incubated for 60 min with serum samples from immunized mice diluted (1:200) in PBS-Tri containing 10% goat serum. After washing three times with PBS/Trit, Cy3-conjugated goat anti-mouse IgG (A10521, Invitrogen) was added as a secondary antibody (1:500 dilution), incubated for 1 h, and washed 5 times. Finally, nuclei were labeled with DAPI/Vectashield (H-1200, Vector Labs, Burlingame, CA, USA). Images were taken using a confocal microscope.