Af647 affinipure goat anti mouse igg h l antibody

mAb binding to whole bacteria was assessed by bacterial flow cytometry, as previously described [34 (link)]. Bacteria were fixed in 4% paraformaldehyde (PFA) for 30 min and resuspended in PBS and stained (10⁶ bacteria/condition) using 5 μM Syto9 (Thermo Fisher Scientific, #S34854) in 0.9% NaCl for 30 min at RT. Bacteria were washed (10 min, 4000 g, 4 °C) and resuspended in PBS, 2% BSA and 0.02% Sodium Azide (PBA). Monoclonal antibodies were pre-diluted in PBA at 20 µg/mL and incubated with bacteria for 30 min at 4 ^°C. Bacteria were washed, and incubated with AF647 AffiniPure goat anti-mouse IgG (H + L) antibody or isotype control (Jackson ImmunoResearch, #115-605-003) for 30 min at 4 ^◦C. After washing, bacteria were resuspended in sterile PBS. Flow cytometry acquisition was performed on a MacsQuant cytometer (Miltenyi) and analyzed on FlowJo software (BD Biosciences). Staining index was calculated by subtracting the Mean Fluorescence Intensity (MFI) of the isotype from the MFI of each condition with the anti-LMW mAbs, then divided by the MFI of the isotype.

The binding of mAbs to whole bacteria was assessed using bacterial flow cytometry assays, as previously described.^{17 (link)} Briefly, fixed C. difficile cells (10^{6 (link)}/condition) were stained with 5 μM SYTO9 dye (Thermo Fisher Scientific, MA, USA) in 0.9% NaCl for 30 min at RT. Bacteria were washed (10 min, 4,000 g, 4°C) and resuspended in 1X PBS, 2% BSA, and 0.02% Sodium Azide (PBA). Mabs were pre-diluted in PBA at 20 µg/mL and incubated for 30 min at 4^◦C. Bacteria were washed, and incubated with AF647 AffiniPure goat anti-mouse IgG (H+L) antibody or isotype control (dilution 1:200, Jackson ImmunoResearch, PA, USA) for 30 min at 4^◦C. After washing, bacteria were resuspended in sterile 1X-PBS. Flow cytometry acquisition was performed on a MacsQuant cytometer (Miltenyi, Germany) and analyzed using FlowJo software (BD Biosciences, CA, USA).