Anti tnf α clone mab11

Purified NK cells were cultured alone or co-cultured with autologous HIV-infected CD4⁺ T cells in complete growth medium in the presence of GolgiStop (BD) and anti-CD107a PE (clone H4A3) antibody (BD) for 16 h. Following incubation, cells were stained with anti-CD3 BV510 (clone UCHT1) (BD), anti-CD56 PerCP Cy5.5 (clone B159) (BD), anti-IFN-γ BV421 (clone B27) (BD) and anti-Siglec-9 PE-Cy7 (clone K8) antibodies. Analysis of Siglec-9⁺ and Siglec-9^- NK cell subsets were made on CD3^- CD56^dim gated cells. For experiments that involve antibody-sialidase conjugates, PBMC were cultured alone or co-cultured with autologous HIV-infected CD4⁺ T cells that had been treated with Sialidase only (300 nM), isotype-matched antibody (300 nM) and indicated amount of antibody or antibody-sialidase conjugate for 2 h at 37°C. Cells were mixed in the complete growth medium in the presence of GolgiStop (BD) and anti-CD107a PE antibody (BD) for 16 h. Following incubation, cells were stained with anti-CD3 BV510 (BD), anti-CD56 PerCP Cy5.5 (BD), anti-IFN-γ BV421 (BD) and anti-TNF-α (Clone MAb11) (BioLegend). NK cells were defined as CD3^- and CD56⁺. Background NK degranulation (PBMC only control) was subtracted. Analysis of CD107a⁺, IFN-γ⁺, and TNF-α⁺ NK cells was made on CD3^- CD56^dim gated cells.

T cell lines were plated at a concentration of 25 × 10⁴ cells/well in 100 μL AIM-V medium (ThermoFisher, Waltham, MA, USA) with 2% human serum in 96-wells plate, and restimulated with either “WT CD4⁺ pool” or “Omicron BA.4/BA.5 CD4⁺ pool” (1 μM/peptide) for 6 h at 37 °C, 5% CO₂. Cells were washed and stained as previously described [22 (link)] with minor modifications as indicated.
Cells were stained intracellularly for anti-CD154 (clone 24–31; Biolegend, San Diego, CA, USA), and anti-IFN-ɣ (clone 4S.B3; BD Bioscience, Allschwil, Switzerland), anti-IL-2 (clone MQ1-17H12; Biolegend), anti-TNF-α (clone Mab11; Biolegend, San Diego, CA, USA), anti-IL-5 (clone TRFK5) and anti-IL-13 (clone JES10-5A2; Both BD Biosciences, Allschwil, Switzerland). Approximately 133,000 events were acquired on a FACS Symphony A3 analyzer (BD Biosciences, Allschwil, Switzerland). FlowJo (version 10, Tree Star, Ashland, OR, USA) was used for flow cytometry data analysis.