Hla dr pe

In 20 CLL cases and 10 healthy volunteers, the CD14⁺CD11b⁺CD15^- monocytes were sorted into HLA-DR^-/low (M-MDSC) and HLA-DR^high (monocytes) fractions. BD FACSAria IIu (detailed configuration in Table S1) (BD Biosciences, Franklin Lakes, NJ, USA) was used for cell sorting. In this case, the samples were labelled with the following monoclonal antibodies: mouse anti-human CD14 FITC (Clone MφP9, Cat No.: 347493), mouse anti-human CD11b PE-Cy7 (Clone ICRF44, Cat No.: 557743), mouse anti-human HLA-DR PE (Clone L243, Cat No.: 307606), and mouse anti-human CD15 APC (Clone HI98, Cat No.:551376) (all from BD Biosciences, Franklin Lakes, NJ, USA; only HLA-DR PE was from BioLegend, San Diego, CA, USA). Cells were incubated for 20 min at room temperature and then washed with PBS; following which, the HLA-DR^-/low and HLA-DR^high populations were sorted (Figure 8A–C).

PBMCs were isolated by Ficoll-Paque (GE Healthcare) and washed with 0.9% NaCl before incubation with 5 µM iron-quercetin (FeQ2) alone and/or in combination with 5 µM apoBet v1 in media containing neither phenol red nor FCS for 18 h as previously described [41 (link)]. Subsequently, cells were stained with combinations of calcein-AM (Thermo-Fisher, Waltham, MA, USA), CD3-APC-Cy7 (eBioscience, clone SK7), CD14-APC-Cy7 (Biolegend, clone M5EZ), HLADR-PE (Biolegend, San Diego, Calif, clone L243PC), and CD86-PE-CY7 (Biolegend, clone IT2.2) for flow cytometric analysis. calcein-AM violet was used as a living marker and to determine the labile iron load in living cells. Doublets were excluded before gating on CD3+ cells in the lymphocyte population or on CD14+ cells in the monocytic population. In monocytic cells this was followed by consecutive gating for HLADR+, CD86+ and calcein+ and geometric mean fluorescence intensity (MFI) was calculated for each fluorochrome. Acquisition and analyses were performed on a FACS Canto II machine (BD Bioscience, San Jose, CA, USA) using the FACSDiva Software 6.0 (BD Biosciences).

The following anti-human antibodies were used for staining: CD68-FITC, CD14-PercP-Cy5.5, HLA-DR-APC, CD15-PE-Cy7, CD11b-PErcP-Cy5.5, CD163-BV421, CD14-APC, HLA-DR-PE, CD33-BV421 and PD-L1-BV421 purchased from Biolegend (San Diego, CA), CD11b-PE, CD3-Alexa Fluor 700, CD19-Alexa Fluor 700, CD19-Alexa Fluor 700, CD163-Alexa Fluor 647, CD16-PE-Cy7, CD3-PE, CD19-PE and CD56-PE purchased from BD Biosciences (San Jose, CA), CD14-PE-TR and CD16 PE-TR purchased from Life Technologies (Carlsbad, CA) and CD86-FITC purchased from R&D systems (Minneapolis, MN). CD32-a-FITC Ab was purchased from Stemcell technologies, (Vancouver, Canada). CD-32-B (F-4) was purchased from Santa Cruz Biotechnology, Santa Cruz, CA, and secondary Ab anti-mouse F(ab’)2 was purchased from Thermo fisher scientific (MA, USA).
Intracellular staining of CD68 was performed as follows: PBMC were stained with surface marker antibodies, fixed with fixation/permeabilization buffer (eBioscience), washed, and stained for intracellular antigens in 1X permeabilization buffer. Cells were analyzed on an LSR Fortessa (BD) flow cytometer, and data analyzed using Flow Jo (Treestar, Ashland, OR). Dead cells were excluded based on viability dye staining (Zombie Aqua Fixable Viability Dye, Biolegend).

PBMCs were obtained from Alternaria-allergic patients after written informed consent. PBMCs were isolated by Ficoll–Paque (GE Healthcare) and washed with 0.9% NaCl before incubation with 5 μM iron quercetin (FeQ2) alone and/or in combination with 2.5 μM apoAlt a 1 in media containing neither phenol red nor FCS for 18 h as previously described [21 (link)]. Subsequently, cells were stained with combinations of CD14-APC-Cy7 (Biolegend, clone M5EZ), HLADR-PE (Biolegend, San Diego, CA, USA, clone L243PC), and CD86-PE-CY7 (Biolegend, clone IT2.2) for flow cytometric analysis. Doublets were excluded before gating on CD14+ cells in the monocytic population, and consecutive gating for HLADR+, CD86+, or HLADR + CD86+. Acquisition and analyses were performed on a FACS Canto II machine (BD Biosciences, San Jose, CA, USA) using the FACSDiva Software 6.0 (BD Biosciences).

Most primary and secondary antibodies came from Abcam: mouse monoclonal CXCL12 (MM0211-9N26), rabbit recombinant monoclonal SCF (EP665Y), rabbit polyclonal HIF-1α (AB103063), or mouse monoclonal connexin-43 (Cx-43) (4E6.2). Goat Anti-Mouse Alexa Fluor^® 488 (IgG H&L) (ab150113), Goat Anti-Rabbit Alexa Fluor^® 405 (IgG H&L) (ab175652), and Donkey Anti-Mouse Alexa Fluor^® 405 (IgG H&L) (ab175658). Rabbit Anti-Human phospho-NF-kB p65 (ser536) (93H1) came from cell signaling. Source for immunophenotyping antibodies: CD34-APC (Clone 581, BioLegend), CD19-FITC (Clone HIB19, BioLegend), CD45-PECy5 (Clone HI30, BioLegend), HLA-DR-PE (clone L243, BioLegend), CD90-APC (5E10, BD), CD73-PE (AD2, BD), and CD105-FITC (266, BD).

Fluorescence-conjugated antibodies were used to stain DCs: ACE2-APC, CD1a-Alexa Fluor 488, CD40-APC, CD80-PerCP/Cy5.5, CD83-PE, CD86-Alexa Fluor 488, C-X-C chemokine receptor type 4 (CxCR4)-PE, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)/CD209-PerCP/Cy5.5, human leukocyte antigen (HLA)-DR-PE, HLA-ABC-APC, and programmed death-ligand 1 (PD-L1)-FITC (all from BioLegend). Isotype-matched antibodies were used as controls. Shortly, DCs were washed and resuspended in phosphate-buffered saline (PBS) + 1% FBS. Then, 3 µL of fluorescence-conjugated antibodies were added to cells and incubated for 30 min, at 4 °C, in the dark. Cells were subsequently washed, resuspended in PBS + 1% FBS, and analysed in an Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA). Data were analysed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA), and the results are presented as mean fluorescence intensity (MFI), obtained by the subtraction of isotype control values.