Facs celesta sorp flow cytometer

To polarize human macrophages, monocyte-derived macrophages were plated in 48-well plates at 5×10⁴ cells per well in 500 µl RPMI medium and allowed to rest for 2 hr at 37°C before treating cytokines or LPS. The human M0 macrophages were either left untreated or treated for 48 hr to induce macrophage polarization: (i) for M1 polarization with 10 ng/ml LPS (E055:B55; Sigma-Aldrich) and 20 ng/ml IFNγ (PeproTech), (ii) for M2 polarization with 50 ng/ml human recombinant M-CSF, 20 ng/ml human recombinant IL-4, and 20 ng/ml human recombinant IL-13 (PeproTech). Human macrophages were then cultured alone or with 0.1 or 1 µg SARS-CoV-2 Spike proteins for 48 hr. The cultured supernatants were collected, and cytokine levels determined using the bead-based immunoassays LEGENDplex Human Macrophage/Microglia Panel (BioLegend). The assays were performed in 96-well plates following the manufacturer’s instructions. For measurements, a FACSCelesta SORP flow cytometer (BD Biosciences) was employed, and data were evaluated with the LEGENDplex Data Analysis software.

To evaluate CD4 T-naïve differentiation, the lymphocyte component of the ascites was analyzed in all culture conditions from 3 patients. Specifically, after 72 h of co-culture with hA-MSCs, the maturation status (naïve, central memory, CM; effector memory, EM; terminal effector memory, TEM) of CD4+ cells was analyzed and compared to the control lymphocytes. We phenotyped T cells using a panel of markers that distinguish lymphocyte cell subsets in T helper CD4+ Th1/Th2/Th17. In detail, we analyzed the CD4+ T cells’ differentiation states using different marker combinations, CD4+ naïve (CD45+CCR7+), CD4+ CM (CD45RA-CCR7+), CD4+ EM (CD45RA-CCR7-), and CD4+ TEM (CD45RA+CCR7-), and the lymphocytes cells subsets in T-helper CD4+ using specific marker combinations, Th1 (CD4+CXCR3+CCR4-), Th2 (CD4+CCR4+CCR6-), and Th17 (CD4+CCR4+CCR6+). The cells were labeled with CD45 APC-Cy7, CD3 BV510, CD4 BV786, CD45RA PE Cy-7, CCR7 BV711, CXCR3 BV421, CCR4 APC, and CCR6 BB515—all from BD Biosciences (BB: Brilliant Blue, BV: Brilliant Violet). Analyses were conducted using a BD FACS Celesta SORP instrument, FACS Celesta SORP flow cytometer, and FACS Diva software version 9.0 (BD Biosciences, CA, USA).

BM Neutrophils (> 95% purity) were resuspended in RPMI medium containing 10% FCS (1 x 10⁶/ml) and incubated for 37°C and 5% CO₂ for 30 min. To induce NETosis, the cells were exposed to the activating agent PMA (10, 30 nM; Sigma-Aldrich) or fMLP (100, 1000 nM; Sigma-Aldrich) for 0-4 hrs at 37°C. NET induction was terminated by 4% PFA for 15 min. Helix NP™ NIR (0.1 µM; Biolegend) and DAPI (0.3 nM; Biolegend) were added to detect NETs. Data acquisition (Helix NP™ NIR⁺ DAPI⁺ Cells) was done on FACSCelesta SORP (BD) flow cytometer and analyzed with FlowJo software (Treestar).

Single cells were re-suspended in 0.1% fatty acid-free BSA-HBSS with 100 μM CaCl₂ and 1 mM MnCl₂ unless otherwise specified. Buffer without divalent cations included 10 mM EDTA. The cells were stained with fluorochrome-conjugated antibodies against various cell surface markers or with different fluorochrome-conjugated proteins, which are listed in the resource and reagent tables in the supplement. LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, LIVE/DEAD Fixable NearIR Dead Cell Stain Kit, or LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Thermo Fisher) were used in all experiments to exclude dead cells. Compensation was performed using AbC Total Antibody Compensation Bead Kit (Thermo Fisher) and ArCTM Amine Reactive Compensation Bead (Thermo Fisher) individually stained with each fluorochrome. Compensation matrices were calculated with FACSdiva software. Data acquisition including cell number count was done on a FACSCelesta SORP (BD) flow cytometer and analyzed with FlowJo 10.8.x software (Tree Star).

Single cells were re-suspended in 0.1% fatty acid free BSA-Hanks’ Balanced Salt Solution (HBSS) with 100 μM CaCl2 and 1 mM MnCl2 unless otherwise specified. Buffer without divalent cations included 10 mM EDTA. The cells were stained with fluorochrome-conjugated antibodies against various cell surface markers or with different fluorochrome-conjugated proteins, which are listed in the resource and reagent tables in the supplement. LIVE/DEAD^™ Fixable Aqua Dead Cell Stain Kit, LIVE/DEAD^™ Fixable NearIR Dead Cell Stain Kit or LIVE/DEAD^™ Fixable Yellow Dead Cell Stain Kit (Thermo Fisher) were used in all experiments to exclude dead cells. Compensation was performed using AbC^™ Total Antibody Compensation Bead Kit (Thermo Fisher) and ArCTM Amine Reactive Compensation Bead (Thermo Fisher) individually stained with each fluorochrome. Compensation matrices were calculated with FACSdiva software. Data acquisition included cell number count was done on a FACSCelesta SORP (BD) flow cytometer and analyzed with FlowJo 10.8.x software (Treestar).