Fluorescein isothiocyanate (fitc)

At least 2 × 10⁵ cells were suspended in 30 μL phosphate-buffered saline (PBS) containing 1% fetal bovine serum and incubated for 20 minutes in the presence of phycoerythrin-labeled antibodies specific for CD29, Sca-1, CD90.2, CD105, CD73, CD117, CD34, CD133, CD45, Gr-1, CD11c, and F4/80, or with fluorescein isothiocyanate-labeled antibodies against CD31 (Beckman Coulter, Fullerton, CA, USA). Nonspecific fluorescence was determined using immunoglobulin G for each antibody isotype (eBioscience, San Diego, CA, USA).

For monitoring the cellular immunity, T cells, B cells, NK cells, Treg cells, and lymphocyte subpopulations were analyzed by a 4-color flow cytometry (EPICS XL, Beckman Coulter Inc., USA). Fluorescein isothiocyanate (FITC)-, PE-Cy5-, PerCP-, allophycocyanin (APC)-, or PE-Texas Red (ECD)-conjugated antibodies against CD3, CD4, CD8, CD56, CD19, CD25, CD28, HLA-DR, CD45RA, and CD45RO were purchased from Beckman Coulter Inc. Cells were labeled according to the manufacturer's protocols.

The Monoclonal antibodies (mABs) used for phenotypic analysis included Fluorescein isothiocyanate (FITC)-, Phycoerythrin (PE)-, Phycoerythrin Cyanine 5.5 and allophycocyanin (APC)-labeled anti-CD45, anti-CD3, anti-CD56, NKp30, NKp44 (Beckman Coulter, Brea, CA, USA), anti-CD16, NKp46, NKG2A (R&D System), anti-CD94, anti-CD69, anti-CD18, anti-CD49, anti-CD62L, anti-CD38 and CXCR4 (BD Pharmingen, San Jose, CA, USA). Phenotype evaluation was performed by direct immunofluorescence according to previously reported methods [29 (link)]. NK cells recovered from immunodeficient NOD/SCID gamma (NSG) mice were stained with anti-mouse CD45-APC-Cy7, anti-human CD45-PE-Cy7, CD3-PerCP-Cy5.5, CD56-FITC (Biolegend, San Diego, CA, USA) and CD94-APC (BD).

Monocyte subsets were analyzed by gating for CD14 (PacificBlue; BD Biosciences) and CD16 (PerCPCy5.5; BD Biosciences) in order to distinguish the three subsets of classical/inflammatory (HLA-DR⁺CD16^-CD14⁺), intermediate (HLA-DR⁺CD16⁺CD14⁺) and nonclassical (HLA-DR⁺CD16⁺⁺CD14^-) monocytes, as described earlier (27 (link)). The gating strategy is depicted in Figure S1F. Sorting of monocyte subsets was performed on a MoFlo Astrios EQ (Beckman Coulter) cell sorter. To characterize iMacs, cells were stained for CD206 (APC, eBioscience), CD45 (PE Beckman-Coulter), CD33 (PerCP5.5, Biolegend), CD14 (FITC, Beckman-Coulter), CD11b (PE BD), or with the corresponding isotype controls and analyzed on an Accuri C6 flow cytometer (BD Bioscience).

RBCs were freshly isolated from all patients and samples were analyzed in parallel. Eryptosis measurements were performed by using AnnexinV. AnnexinV is used to identify eryptotic cells [19 (link),20 (link)] that are avidly bound by phosphatidylserine (PS). Flow cytometric analyses were used to measure AnnexinV bound to PS exposed on RBC surfaces. Briefly, RBCs separated from leukocytes were washed in 5 mM CaCl2 Ringer solution (1 μL of leukocyte in 400 μL of Ringer solution) and then stained for 20 min, avoiding light, with 1 μL of AnnexinV conjugated with FITC (Beckman Coulter, Brea, CA, USA). Before analysis with Navios Flow Cytometer (Beckman Coulter, Brea, CA, USA), 400 μL of Ringer solution was added. This led to an evaluation of the subpopulations of eryptotic RBCs over 1 h. RBCs that had exposed PS at their surface were identified as gating and enumerating RBCs. After that, FSC (forward scatter) was assessed and AnnexinV fluorescent intensity was measured, with an 488 nm excitation wavelength and an 530 nm emission wavelength. A minimum of 100,000 events were collected on each sample.

Intracellular staining of WASp using phycoerythrin (PE)-labeled anti human WASp antibody (sc-13139PE (clone: B-9), Santa Cruz Biotechnology, United States), was carried out on non-erythroid blood cells in peripheral blood. Cells were gated using side scatter vs CD45 labeled with fluorescein isothiocyanate (FITC) (A07782; Beckman Coulter Life Sciences, United States). Cells were acquired on an in-house flow cytometer (Navios™ EX System, Beckman Coulter, United States). Median fluorescence intensity (MFI) and Stain Index (SI) in stimulated and unstimulated samples were calculated.

After trypsinization, single cell suspension (1 × 10⁷) prepared in regular medium was incubated with an anti-CD44 antibody conjugated with FITC (Beckman Coulter Inc., Krefeld, Germany, IM1219U). CD44⁺ cells sorting was performed on a BD FACSAria (BD Biosciences Pharmingen, San Diego, CA, USA) using the index sorting function.