Anti tnf α mab11

Cell surface marker expression was analyzed on a Becton Dickinson LSR Fortessa^TM flow cytometer after staining with fluorochrome-conjugated antibodies. Intracellular staining was performed using the fix/perm buffer set (Biolegend), according to the manufacturer's instructions. Antibodies used were conjugated anti-NKp30 (P30–15, Biolegend), anti-NKp44 (P44–8.1, BD), anti-NKp46 (29A1.4, Biolegend), anti-IFN-γ (485.B3, Biolegend), anti-TNF-α (MAb11, eBioscience), anti-RANKL (MIH24, Biolegend), anti-Ki-67 (16A8, Biolegend), anti-CD107a (H4A3, BD), anti-CD57 (HCD57, Biolegend), anti-CD56 (N901, Beckman Coulter), anti-CD14 (HCD14, Biolegend), anti-CD16 (3G8, Biolegend), and anti-CD3 (UCHT1, Biolegend). Annexin V/7-AAD staining was performed according to the manufacturer's protocol (Biolegend). The data were analyzed using FlowJo v10.2 software.

Fluorochrome-labeled antibodies used for flow cytometric analyses were obtained from the following vendors: anti-CD3 (UCHT1), anti-CD16 (eBioCB16), anti-CD56 (CMSSB), anti-CD107a (eBioH4A3), anti-CD107b (eBioH4B4), anti-CD300c (TX45), anti-TNFα (MAb11), anti-MIP1α (PFFM3) and anti-CD69 (FN50) were from eBiosciences and anti-IFN-γ (B27) was from BD Biosciences. Purified antibodies anti-human IL-2 (AB12-3G4), mouse IgG2a κ isotype (eBM2a), mouse IgG1 κ (MOPC-21) and anti-CD300c (TX45) were obtained from eBiosciences. For the real time PCR experiments, primers for CD300A, CD300C and 18sRNA genes were purchased from SA Biosciences. STAT5 inhibitor N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide was purchased from Calbiochem/EMD Millipore. Recombinant IL-2 was obtained from National Cancer Institute, Frederick, MD. Cytokines IL-12, IL-15 and IL-4 are from R&D Systems, IL-18 is from Medical and Biological Laboratories (MBL) and IL-21 is from Peprotech. Phospholipids 1-palmitoyl-2-oleoyl (PO) phosphatidylserine (POPS) (PS), phosphatidylethanolamine (POPE) (PE) and phosphatidylcholine (POPC) (PC) were purchased from Avanti Polar Lipids.

PBMC were stained for extracellular markers (clones in parenthesis) with anti-CD2 (RPA-2.10, BioLegend, San Diego, CA, USA), anti-CD3 (BW264/56), anti-CD16 (3G8), anti-CD56 (REA196), anti-CD57 (TB03), anti-NKG2A (REA110), all from Miltenyi Biotec, Auburn, CA, USA and anti-NKG2C (134591, R&D Systems, Minneapolis, MN, USA). LIVE/DEAD Fixable Far Red (Invitrogen) was used to exclude dead cells. Fixation and permeabilization were performed using MACS Inside Stain Kit (Miltenyi Biotec) with polyclonal goat anti-human FcεRIγ (EMD Millipore, Etobicoke, ON, Canada), anti-IFN-γ (4S.B3), and anti-TNF-α (MAb11) from eBioscience, San Diego, CA, USA added for intracellular staining. The fluorescence minus one strategy was used to adjust multicolor compensation. Phenotypic analysis was performed using Kaluza Flow Cytometry Software 1.2 after data acquisition on a MoFlo Astrios EQ flow cytometer (both Beckman Coulter, Brea, CA, USA).