Cd38 pe cy7

The CLL cells from all the blood samples were stained with optimal concentrations of antibody combinations and directed against the following surface antigens: CD183(CXCR3)-FITC, CD20-PE, CD5-PerCP-Cy5.5, CD38-Pe-Cy7, CD49d-APC, CD19-APC-Cy7, CD184 (CXCR4)-BV421 and HLA-DR-BV510 (all procured from BioLegend), as previously reported [5 (link),15 (link)]. Isotype-matched antibodies (BioLegend) were used as negative controls.
The determination of s-CLL and l-CLL cells was conducted using FSC data and a back-gating strategy. The analysis was performed using a BD FACSCanto II (Becton Dickinson) instrument, and data acquisition was performed using BD FACSDiva software (v.8.0.2; Becton Dickinson). Flow cytometry data were analysed using FlowJo v.X0.7 software (Tree Star, Inc., San Carlos, CA, USA). In all the experiments, a minimum of 10,000 events was counted. The results were expressed as a percentage and mean fluorescence intensity (MFI).

SP of MM cells was evidenced by DCV staining (5 μM, Thermo Fisher Scientific, Waltham, MA, USA) for 90 min at 37 °C in the absence or presence of the ABC transporter inhibitor reserpine (50 μM, Sigma-Aldrich, St. Louis, MO, USA). SP analysis of MM cells was performed by flow cytometry. Fresh bone marrow samples were also stained with CD138-FITC (Biolegend, San Diego, CA, USA) and CD38-PE/Cy7 (Biolegend, San Diego, CA, USA) to identify MM plasma cells. DCV was excited at 405 nm and its emission fluorescence detected using 450/50 nm (DCV blue) and 675/20 (DCV red) band pass filter systems. To determine if SP cells exhibit clonogenic properties, colony forming assays were also performed after sorting of SP cells. SP cells were seeded in triplicate at 300 cells/ml and incubated as described above.

Tumor-infiltrating lymphocyte isolation was performed as previously described.9 (link) Cells were blocked with antimouse CD16/32 for 30 min at 4°C and then stained with CD45 APC (Cat #187357), CD4 APC-Fire 750 (Cat #244105), CD8 PercpCy5.5 (Cat #277115), Foxp3 Alexa488 (Cat #227489), Granzyme B Pacific-blue (Cat #267707), Gr1 BV510 (Cat #238839), CD11b Alexa700 (Cat #259438), CD38 PE-Cy7 (Cat #216741), and CD206 PE (Cat #225355) from BioLegend. Samples were run on a Gallios (BD Biosciences) flow cytometer and analyzed with Kaluza Analysis Software.

Purified BMMCs of the six COVID-19 patients were used to stain the HSPCs and flow cytometry was performed on FACSymphony^TM S6 (BD Biosciences). The following antibodies for the HSPCs were used: lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56/Pacific Blue) (Biolegend, clone UCHT1; HCD14, 3G8, HIB19, 2H7, HCD56, Cat. no. 348805, RRID: AB_2889063), CD34/APC (BD Biosciences, clone 581, Cat. no. 555824, RRID: AB_398614), CD38/PE-Cy7 (Biolegend, clone HIT2, Cat. no. 303516, RRID: AB_2072782), CD90/PerCP-Cy5.5 (BD Biosciences, clone 5E10, Cat. no. 561557, RRID: AB_10712762), CD45RA/APC-Cy7 (Biolegend, clone HI100, Cat. no. 304128, RRID: AB_10708880), CD49f/BV605 (BD Biosciences, clone GoH3, Cat. no. 740416, RRID: AB_2740146), CD10/BV786 (BD Biosciences, clone HI10a, Cat. no. 564960, RRID: AB_2739025), LIVE/DEAD dye/BV510 (Invitrogen, Cat. no. L34957), and Annexin V/FITC (BD Biosciences, Cat. no. 556547, RRID: AB_2869082). Gatings included HSC (Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺CD10⁻), MPP (Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁻CD10⁻).

Donor PBMCs was stained with live-dead marker (1:1000; Invitrogen, LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit) to gate out the dead cells for 20 min in 4 °C. After washing with FACS buffer, the cells were stained with respective B, T cell marker panel antibodies diluted in FACS buffer (1xPBS, 2% FBS, 0.1% sodium azide) at 4 °C for 30 min. B cell panel: CD19-BV510 (1:40; Biolegend, Cat. No. 363020), CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD3-APC-Cy7 (1:125; Biolegend, Cat. No. 317342), CD10-PE-Dazzle594 (1:50; Biolegend, Cat. No. 312228), CD21-APC (1:50; Biolegend, Cat. No. 354906), CD27-BV650 (1:40; Biolegend, Cat. No. 302828), CD38-PE-Cy7 (1:40; Biolegend, Cat. No. 356608), PD-1-BV711 (1:20; Biolegend, Cat. No. 329928), IgD-BUV 737 (1:40; BD, Cat. No. 612798), IgG-PerCP (1:40; BD), IgA-PE (1:40; Miltenyi, Cat. No. 130-114-002). T cell panel: CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD19-APC-Cy7 (1:50; BD, Cat. No. 557791), CD3-BUV395 (1:40; BD, Cat. No. 563546), CD4-BUV496 (1:40; BD, Cat. No. 612936), CD8-PE (1:40; BD, Cat. No. 555367), CXCR5-PE-Cy7 (1:100; BD, Cat. No. 624052), CD25-BV421 (1:40; BD, Cat. No. 562442), CD127-FITC (1:20; BD, Cat. No. 557938). The samples were then washed, resuspended in FACS buffer and analysed on BD LSRFortessa flow cytometer (Data availability—Figshare 10.6084/m9.figshare.23549964).

Antibodies with the following specificities used for flow cytometry and histology were purchased from BD Bioscience, eBioscience or Biolegend: CD45.2-biotin, Lamda Light chain-biotin, GFP- Alexa Fluor 488 (AL488), rabbit IgG-PE, CD38-PE-Cy7, CD95-PE-Cy7, BCL6-Al647, Streptavidin-Al700 or -Brilliant Violet 421 (BV421), CD45RA (B220)-allophycocyanin (APC)-Cy7, IRF4-eFluor 450. RelB (C19) was from Santa Cruz Biotech, rabbit IgG Fab₂-Al555 was from Cell Signaling Technologies. Purified αCD40 (FGK4.5) (RRID: AB_2490239) and αCD40L (MR1) (RRID: AB_1612465) were purchased from BioXCell. AFRC-Mac-1 cell (glycoprotein of dog chlamydomonas) (RRID: CVCL_K178) (Sigma-Aldrich) produced Rat IgG2a istotype control was purified from culture supernatants by affinity chromatography, using a staphylococcal protein G column (ThermoFisher Scientific) and filter sterilized. Hamster IgG was from Jackson ImmunoResearch Laboratories.

Tumor cells and T cells were phenotyping with CD3‐APC (OKT3, UCH1), CD19‐APC CY7 (SJ26C1), CD4‐PE Cy7 (RPA‐T4, A161A1), CD8‐pacific blue (RPA‐T8, SK1), CD10‐APC (HI10a), CD22‐PE (HIB22), CD38‐PECy7 (HTT2), CD56‐PE CY7 (MEM‐188), CD62L‐BV510 (DREG‐56), PDL1‐PE (29E.2A3), PD1‐FITC (EH12.2H7), streptavidin‐APC‐Cy7, and 7AAD, which were purchased from BioLegend. CD20‐FITC (2H7), CD45RA‐FITC (HI100), and CD62L‐PE (DREG‐56) were purchased from BD. CD137‐PE, CD34‐APC, and antibiotin‐PE were purchased from MACS. The percentage of CAR‐CD19 was determined by biotinylated Erbitux. All cells were analyzed MACSquant with a filter set for eight fluorescence signals and analyzed with FlowJo software (Tree Star).