Anti cd15 bv605

The method was adapted from Hartung et al. (Cytometry A. 2019 Mar;95(3):332-338). Briefly, erythrocytes were depleted from healthy donors’ blood by osmotic shock using an Erythrocyte Lysis Buffer (0.15 M NH₄Cl; 10 mM NH₄Cl; 0.1 mM EDTA). Human leukocytes were seeded in a 24-well plate at a density of 1x10⁶ cells per well and cultured in RPMI 1640 (Lonza) supplemented with 10% heat-inactivated FBS (Gibco). The obtained leukocytes were incubated with fluorescein isothiocyanate (FITC)-labeled (100µg/mL) spores at MOI 5 for 30, 60, and 120 minutes at 37 °C and 5% CO₂. Consequently, leukocytes were harvested with PBS-2mM EDTA and stained with anti-CD45-BUV395 (Biosciences), anti-CD15-BV605 (Biolegend), anti-CD14-PECy7 (Biolegend), and anti-FITC-APC (Invitrogen) for 20 minutes at room temperature (RT). Phagocytosis was assessed by using an LSR Fortessa (Becton Dickinson) flow cytometer and data were analyzed with FlowJo v10.6.2 software.

Fresh whole-blood leukocytes were analyzed by flow cytometry to identify and quantify the major leukocyte populations. Briefly, 200 μl EDTA-stabilized venous blood was stained with a panel of fluorochrome-conjugated antibodies (anti-CD45-PerCP [Clone: HI30; BioLegend], anti-CD3-APC/Cy7 [Clone: OKT3; BioLegend], anti-CD4-BUV395 [Clone: SK3; BD Biosciences], anti-CD8-BUV737 [Clone: SK1; BD Biosciences], anti-CD19-PE [Clone: HIB19; BioLegend], anti-CD14-Alexa Fluor 488 [Clone: HCD14; BioLegend], anti-CD16-PE/Cy7 [Clone: 3G8; BioLegend], anti-CD15-BV605 [Clone: W6D3; BioLegend], and anti-CD56-APC [Clone: HCD56; BioLegend]) for 15 min at room temperature in BD Truecount tubes. Red blood cells were lysed (BD FACS Lysing solution), and leukocytes were analyzed immediately on a BD Fortessa flow cytometer with BD FACSDiva software. Absolute cell numbers were calculated according to the manufacturer’s instructions (Truecount Tubes, BD Biosciences).
For the characterization of both rare and abundant leukocyte populations in the blood, cryopreserved PBMCs (4–8 × 10⁵ cells per individual) from the PD-L1-deficient siblings, their heterozygous mother, a healthy adult, and age-matched controls, and the previously described PD-1-deficient child (Ogishi et al., 2021 (link)) were analyzed by spectral flow cytometry as previously described (Ogishi et al., 2023 (link)).