Mouse seroblock fcr

For membrane CD14 flow cytometry, BMDMs seeded in 24-well plates were washed and incubated for 30 min at 37°C in E-total buffer supplemented with or without 5 mM of ATP, in presence or absence of P2X7 receptor antagonist A438079 (10 μM). To stain surface CD14, cells were washed and incubated with mouse seroblock FcR (BD biosciences) and then stained with anti-mouse CD14 (clone rmC5-3; 553738; BD biosciences; RRID:AB_395020) for 30 min at 4°C. Cells were washed again and incubated with secondary Alexa Fluor 647 goat anti-rat IgG (H+L) (A21247; Invitrogen, RRID:AB_141778) for an additional 30 min at 4°C. Finally, cells were washed and fixed with 4% PFA in PBS and then scrapped and aliquoted in flow cytometry tubes. For human P2X7 flow cytometry, monocytes were determined from peripheral blood mononuclear cells from non-septic and septic patients by CD3⁻ CD14⁺ selection, and P2X7 receptor surface expression was determined using the monoclonal anti-P2X7 L4 clone (Buell et al., 1998 (link); Martínez-García et al., 2019 (link)). All samples were subjected to flow cytometry analysis using a BD FACSCanto flow cytometer (BD) and FACSDiva software (BD, RRID:SCR_001456) by gating for BMDM cells based on FSC versus SSC parameters.

4x10⁶ splenocytes were isolated and cultured with 5 μg of recombinant protein myosin for 12 h at 37°C in CO₂ incubator followed by re-incubation with 10 μg/ml of Brefeldin A (BD Biosciences) in dark for 6 h at 37°C in CO₂ incubator. Cells were washed and incubated with mouse seroblock FcR (BD Biosciences) for 10 min to block the non-specific binding of antibodies to CD16 and CD32 markers to curtail background signal. Cells were re-incubated in dark at 4°C overnight with FITC-CD4 antibody, washed with PBS, fixed and permeabilized for 15 min. Cell suspension was divided in separate tubes and incubated in presence of PE labelled anti-mouse monoclonal antibodies to IL-2, (clone-JES6-5H4), IL-4 (clone-BVD4-1D11), IFN-γ (clone-XMG1.2) and IL-10 (clone-JES5-16E3). Data was acquired on FACS Calibur and analyzed as above. PE labelled anti-mouse isotype control antibodies (IgG2b k for IL-2, IL-4, IL10 and IgG1 k for IFN-γ; BD Biosciences) were used as negative control.