Anti cd14 apc cy7

Uninfected CD4 T cells were isolated as above and treated or not with HDACi at the indicated doses. Meanwhile NK cells were isolated as above. NK and CD4 T cells were cultured at a 1:1, 1:0.2, or 1:0.01 ratio for 5 hours at 37°C in the presence of an anti-CD107a PE-Cy7 antibody (Biolegend). Experiments using K562 cells were performed at a 1:1 ratio for 5 hours as with CD4 T cells. For both CD4 T and K562 experiments, cells were then washed and stained with a panel of: LIVE/DEAD fixable near-IR dead cell stain kit (Life Technologies), anti-CD3 eflour450 (eBioscience), anti-CD56 APC (Miltenyi), anti-CD16 FITC (Biolegend), anti-CD14 APC-Cy7 (Biolegend), anti-CD19 APC Cy7 (Biolegend). NK cells were gated on APC Cy7 negative cells (live, CD14-, CD19-), CD3 negative, CD56 positive cells.

PBMC were cultured for 24h in the absence or presence of 100nM panobinostat, 20nM panobinostat, or 10nM romidepsin. After culture, wells were stained with a panel of: LIVE/DEAD fixable near-IR dead cell stain kit (Life Technologies), anti-CD3 eflour450 (eBioscience), anti-CD16 APC (Cambridge Bioscience), anti-CD56 FITC (Cambridge Bioscience), anti-CD14 APC-Cy7 (Biolegend), anti-CD19 APC Cy7 (Biolegend), NKG2D PECy7 (Cambridge Bioscience), and CD335 (NKp46) PE (Miltenyi Biotec). NK cells were gated on APC Cy7 negative cells (live, CD14-, CD19), CD3 negative, CD56 positive cells.

ROS production was quantified by flow cytometry using the Phagoburst Kit (Glycotope Biotechnology GmbH) according to manufacturer’s instructions. Briefly, heparinized cord blood was incubated on ice with anti-CD14 APC/Cy7, anti-CD16 APC and anti-CD62L Brilliant Violet 421 antibodies (Biolegend). For detailed gating strategy see Additional file 3: Figure S2. Cells either remained unstimulated or were incubated with unlabeled opsonized E. coli (0.9–1.8 × 10⁸/ml), phorbol 12-myristate 13-acetate (PMA) (0.74 μM), or N-formyl-methionyl-leucyl-phenylalanine (fMLP) (0.45 μM) as stimulants for 10 min at 37 °C; subsequently, DHR was added for 10 min which—by ROS-dependent conversion into rhodamine 123—allowed the quantification of reactive oxidants and determination of the percentage of phagocytes that produced ROS. The ROS production per cell was quantified by MFI. Kit-included DNA-Dye was used after red blood cell lysis using PFA-containing BD FACS™ Lysing Solution to differentiate between E. coli and cells. The flow cytometry results were evaluated with FlowJo Software 10.3 (Tree Star Inc., Ashland, OR). Gating of subpopulation was done as described above (Additional file 2: Figure S1 A/B; Additional file 3: Figure S2).

Cells were stained using two different panels with Zombie NIR Fixable Viability stain (BioLegend, San Diego, USA) and combinations of the following fluorochrome-conjugated surface antibodies: CD4 (clone SK3), CD45 (clone HI30), CD56 (clone NCAM16.2), TCR-γ/δ (clone 11F2) all BD Biosciences, Heidelberg, Germany and CD8 (clone RPA-T8), HLA-DR (clone L243), CD45RA (clone HIT100), CD196 (CCR6) (clone G034E3), CD194 (CCR4 clone L29144), CD197 (CCR7) ((clone G043H7), CD57 (clone HNK-1), CD183 (CXCR3) (clone G025H7), CD38 (clone HIT2), CD161 (clone HP-3G10), CD25 (clone M-A251), CD3 (clone UCHT1), CD127 (clone A019D5), CD14 (clone HCD14), CD19 (clone HIB19), CD16 (clone 3G8), TCR Vα7.2 (clone 3C10), TCR Vα24-Jα18 (clone 6B11), CD314 (NKG2D) (clone 1D11), CD4 (clone SK3), TCR Vδ2-FITC, (clone B6), CD39-PE/Cy7 (clone A1) all BioLegend, San Diego, USA. Single-stained Comp Beads (Anti-Mouse Ig,κ/Negative Control Compensation Particles Set, BD Biosciences) were used for compensation. For live/dead compensation, Comp Beads stained with anti-CD14 (APC Cy-7, BioLegend) were applied. The exact composition of these two panels is displayed in S1 Table. All samples were run on a BD LSR Fortessa flow cytometer with FACS Diva version 8 (BD Biosciences) on a PC.

Tonsil mononuclear cells were thawed, washed, and counted as described above. To stain apoptotic and necrotic cells, cells were incubated with Zombie NIR fixable viability dye (Biolegend) for 15 min at room temperature. Cells were washed, resuspended in a surface stain cocktail containing anti-IgD-FITC, anti-CD38-PE, anti-CD27-PE/Cy7, anti-CD19-APC, anti-CD3-APC/Cy7, and anti-CD14-APC/Cy7 (all from Biolegend), and incubated for 45 min on ice. Subsequently, cells were washed two times, passed through a 35 µm nylon mesh and sorted on a BD FACS Aria II at the Harvard Microbiology and Immunobiology (MBIB) Flow Cytometry Core. Lymphocytes were electronically gated according to their forward scatter and side scatter properties, negatively gated to exclude viability dye⁺, CD3⁺, and CD14⁺ cells, and positively gated for B cells (CD19⁺). B cell subsets were subsequently gated as follows: IgD ⁺CD27^- cells (Naive); IgD^- CD38^hi CD27^+/- (GC); IgD^- CD38^lo/mid CD27 ⁺ (Memory) (See also Fig. 1b and Supplementary Fig. 1). The CD27 gate was set based on a PE/Cy7 FMO gating control. Sorted cells were pelleted, washed 2× with PBS, then lysed in Buffer RLT (Qiagen, for RNA analysis), 2% NP-40 lysis buffer (for western blot), or snap frozen in an isopropanol/dry ice slurry (for glycomic analysis).