Cells were isolated from peripheral blood using Ficoll density gradient centrifugation and perioheral blood mononuclear cells (PBMC) were stained with the following anti-human antibody staining reagents: CD3-BV711 (clone: HIT3a, BD Biosciences); CD3-PE-Cy5.5 (clone: 7D6, Fisher Scientific); CD14-BV711 (clone: M5E2, BD Biosciences); CD14-PE-Cy5.5 (clone: TuK4, Thermo Fisher Scientific); IgD-FITC (IA6-2, BD Biosciences); CD19-PE-Cy7(clone: SJ25C1, BD Biosciences); CD27-APC-eFluor780 (clone: O323, Fisher Scientific); CD38-V450 (clone: HIT2, BD Biosciences); CD138-APC (clone: 44F9, Miltenyi Biotec); CXCR4-PE (clone: 12G5, BioLegend); CXCR4-BV711 (clone:12G5, BD Biosciences); CXCR3-PE(G025H7, BioLegend); Blimp1-PE (6D3, BD Biosciences); BCMA-PE (19F2, BioLegend), IL-6R-PE (M5, BD Biosciences). Approximately, 1 × 103 to 5 × 103 were collected for each cell population by using FACSAria II (BD Biosciences).
Cd38 v450
Manufactured by BD
The CD38 V450 is a fluorescent dye-conjugated antibody used for the identification and analysis of CD38-positive cells in flow cytometry applications. It provides a specific and reliable method for detecting the presence of the CD38 protein on the surface of cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd19 pe cy7, by BD (3 mentions)
Facsaria 2, by BD (3 mentions)
Cd45 v500, by BD (3 mentions)
Cd138 apc, by Miltenyi Biotec (2 mentions)
Cd3 bv711, by BD (1 mentions)
Cd14 bv711, by BD (1 mentions)
Igd fitc ia6 2, by BD (1 mentions)
Cxcr4 pe, by BioLegend (1 mentions)
Cxcr3 pe, by BioLegend (1 mentions)
Blimp1 pe, by BD (1 mentions)
Bcma pe, by BioLegend (1 mentions)
Live dead, by Thermo Fisher Scientific (1 mentions)
Cd14 bv711, by BioLegend (1 mentions)
Cd3 bv711, by BioLegend (1 mentions)
Lymphocyte separation medium, by Corning (1 mentions)
Cd10 apc, by BD (1 mentions)
Cd34 percp, by BD (1 mentions)
Cd19 apc h7, by BD (1 mentions)
Cd20 pe cy7, by BD (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
7 protocols using cd38 v450
1
Multiparametric Phenotyping of Human Blood Cells
2
Isolation and Purification of Antibody-Secreting Cells
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PBMCs and BM mononuclear cells were isolated from fresh blood and BM aspirate samples, respectively, by density gradient centrifugation using Lymphocyte Separation Medium (Cellgro/Corning). After isolation, the mononuclear cell fractions were further enriched by negative selection using a MACS column that removed CD3+/CD14+ cells, a commercial human pan-B cell enrichment kit that removes cells expressing CD2, CD3, CD14, CD16, CD36, CD42b, CD56, CD66b, CD123, and glycophorin A, or a custom stem cell kit that removes CD66b+/GPA+/CD3+/CD14+ cells (StemCell Technologies). Enriched fractions were then stained using the fluorescently conjugated antibodies IgD–FITC (Cat. no. BD555778; BD Biosciences), CD3-BV711 (Cat. no. 317328; BioLegend), CD14-BV711 (Cat. no. 301838; BioLegend), CD19-PE-Cy7 (Cat. no. 301838; BD Biosciences), CD38-V450 (Cat. no. BDB561378; BD Biosciences), CD138-APC (Cat. no. 130-117-395; Miltenyi Biotech), CD27-APC-e780 (Cat. no. 5016160; eBiosciences), and LiveDead (L34966; Invitrogen) and populations of ASC were purified by fluorescent activated cell sorting for further experiments. Cell sorting experiments were performed on a FACS Aria II (BD Biosciences) using a standardized sorting procedure that used rainbow calibration particles to ensure consistency of sorts over time. The gating strategies used to isolate ASC populations are in Figs S1 and S6 .
3
Multiparameter Flow Cytometry for B-ALL MRD Detection
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MPFC was performed with a panel of antibodies designed for B‐ALL. The diagnostic panel for B‐ALL contained CD45, CD7, CD19, CD13, CD33, CD34, CD117, HLA‐DR, CD10, cMPO, cCD3 and cCD79a and its extended panel included CD66c, CD22, CD20, CD58, CD38, CD123, CD45, cytoplasmic heavy chain of immunoglobulin M(cu) and cytoplasmic TDT (BD Bioscience; San Jose, CA; Beckman‐Counter; Indianapolis, IN).
15 (link) FACS Aria II (BD Biosciences) with Diva program was used to acquire and analyze the data. MRD panel included CD58‐FITC, KORSA‐PE, CD34‐Percp, CD20‐PE‐cy7, CD10‐APC, CD19‐APC‐H7, CD38‐V450 and CD45‐V500 (BD Bioscience and Beckman‐Counter).
Negative MRD by MPFC was defined as <10−4 blasts (0.01%) in bone marrow samples and Flow‐MRD positivity was defined as >10−4 blasts (0.01%) in bone marrow. In our study, patients with sustained undetectable MRD were defined according to negative MRD results observed at the end of consolidation and any of the previous time points.
15 (link) FACS Aria II (BD Biosciences) with Diva program was used to acquire and analyze the data. MRD panel included CD58‐FITC, KORSA‐PE, CD34‐Percp, CD20‐PE‐cy7, CD10‐APC, CD19‐APC‐H7, CD38‐V450 and CD45‐V500 (BD Bioscience and Beckman‐Counter).
Negative MRD by MPFC was defined as <10−4 blasts (0.01%) in bone marrow samples and Flow‐MRD positivity was defined as >10−4 blasts (0.01%) in bone marrow. In our study, patients with sustained undetectable MRD were defined according to negative MRD results observed at the end of consolidation and any of the previous time points.
4
Comprehensive B Cell Profiling in Blood
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B cell differentiation in peripheral blood was detected in 100 μl of blood samples washed twice with staining buffer (PBS containing 1% BSA) and stained with a cocktail of mAbs: CD45 V500, CD19 PE-Cy7, CD21 APC, IgM FITC, IgD PE, CD27 PerCP-Cy5.5, and CD38 V450 (all from BD Bioscience) for 20 minutes in the dark at room temperature. Erythrocytes were lysed with BD FACS Lysing solution (BD Bioscience) and washed twice with buffer. B cells were then classified according to their maturation stage into (i) immature (CD21-CD27-) B cells, (ii) naive (CD21+CD27-IgM+) B cells, (iii) memory B cells (CD27+CD19+), (iv) unswitched memory B cells (CD27+IgM+IgD+), (v) memory-switched B cells (CD27+IgM-IgD-), (vi) transitional B cells (CD38+IgM+), and (vii) plasmablasts (CD38+IgM-) (23 (link)) For gating strategy see Supplementary Figure S3 Table S1
5
Immunohistochemistry and Flow Cytometry Analysis
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Immunohistochemistry. Immunohistochemical analysis was performed on 4 μm, formalin-fixed, paraffin-embedded sections in all cases. A broad immunohistochemical panel was utilized in the evaluation of the cases. Primary antibodies included CD34 (QBEnd/10, Leica (Novocastra)), CD117 (YR145, Cell Marque), CD71 (MRQ-48, Cell Marque), E-Cadherin (4A2C7, Life Technologies), Myeloperoxidase (Dako), Hemoglobin (Cell Marque), Glycophorin A (GA-R2, Ventana), CD61 and TP53 (DO-7, Ventana).
Flow cytometry. After isotonic erythrocyte lysis, flow cytometric immunophenotyping was performed on anticoagulated bone marrow aspirate specimens using previously described methods [10 (link)]. Samples were examined with flow cytometric immunophenotyping using two eight-color tubes containing antibodies from BD Biosciences (Tube1: CD13 PE, CD15 V450, CD16 APC-H7, CD33 PE-Cy7, CD34 PerCP-Cy5.5, CD45 V500, CD117 APC, HLA-DR FITC and Tube2: CD2 FITC, CD7 PE, CD34 PerCP-Cy5.5, CD36 APC, CD38 V450, CD45 V500, CD56 PE-Cy7, CD64 APC-H7). A total of 100, 000 events were collected per case. The data were analyzed using Kaluza software (Beckman-Coulter, Brea, CA) and/or Diva software (BD Biosciences).
6
Lymphocyte Subset Analysis by Flow Cytometry
7
Quantifying Immune Cell Activation
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To quantify the level of activation, proliferation and cell death, cryopreserved CD4 subsets remaining from the sort were thawed. Approximately 2 million cells were stimulated with either DMSO (Sigma Aldrich, Cat no D2650-5X10ML) or PMA/ionomycin (final concentration of 10nM PMA and 0.5μM ionomycin) for 72 h. After the stimulation, 1.5 million cells were washed and surface stained for live/dead aqua, CD69 Alexa Fluor 647 (Biolegend, Cat no 310918, clone FN50), HLA-DR APC-Fire750 (Biolegend, Cat no 307658, clone L243), and CD38 V450 (BD biosciences, Cat no 561378, clone HIT2). Cells were then permeabilized using BD Cytofix/Cytoperm buffer (BD Biosciences, Cat no 554714) and stained intracellularly with Ki-67 PE (BD biosciences, Cat no 556027, clone B56). Samples were run within 2 h of staining completion on BD LSR Fortessa flow cytometer and the data was analyzed using FlowJo version 10.
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