The following monoclonal Abs were used for flow cytometry and cell sorting: anti-CD34 (581, BD Biosciences, 1:5), -CD38 (HIT2, BD Biosciences, 1:5), -CD45RA (HI100, BD Biosciences, 1:20), -CD90 (5E10, BD Biosciences, 1:20), -CD49f (GoH3, Biolegend, 1:5), and -CD45 (HI30, BD Biosciences, 1:20). A mixture of biotin-conjugated anti-CD2, -CD3, -CD11b, -CD14, -CD15, -CD16, -CD19, -CD56, -CD123, and -CD235a (130-092-211, Lineage Cell Depletion Kit human, Miltenyi Biotec Inc., 1:5) was used as the lineage mix. The following Abs were used for immunocytochemistry: anti-human TRF1 (TRF-78, ab10579, Abcam, 1:100), 53BP1 (NB100-304, 1:200), and RPA32 (sc-28709, 1:200).
Cd38 hit2
Manufactured by BD
CD38 (HIT2) is a laboratory equipment product used for cell surface antigen detection. It functions as a receptor involved in cell adhesion, signal transduction, and calcium signaling. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (2 mentions)
Cd19 sj25c1, by BD (2 mentions)
Cd8 sk1, by BD (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Cd3 sk7, by BD (2 mentions)
Igd ia6 2, by BD (2 mentions)
Cd62l dreg 56, by BD (2 mentions)
Cd45ra hi100, by BD (2 mentions)
Cd31 wm59, by Thermo Fisher Scientific (2 mentions)
Cd45ro uchl1, by BD (2 mentions)
Cd21 b ly4, by BD (2 mentions)
Igm mhm 88, by BioLegend (2 mentions)
Cd10 hi10a, by BD (2 mentions)
Cd138 b b4, by Bio-Rad (2 mentions)
Iga g20 359, by BD (2 mentions)
γδ tcr b1, by Thermo Fisher Scientific (2 mentions)
αβ tcr ip26, by Thermo Fisher Scientific (2 mentions)
Cd57 nk 1, by BD (2 mentions)
Foxp3 pch101, by Thermo Fisher Scientific (2 mentions)
Cd90 5e10, by BD (1 mentions)
8 protocols using cd38 hit2
1
Multiparameter Flow Cytometry and Cell Sorting
2
Antigen-Specific T Cell Activation Analysis
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Cryopreserved PBMCs were thawed and 106 live cells were plated out in 96 well U-bottom plates. Spike protein peptide pool (GenScript, New Jersey, USA), or Chimpanzee Adenovirus Y25 hexon protein peptide pool (JPT Peptide Technologies, Berlin, Germany) was added to each well at 1 μg/mL and cells incubated for 20–24 h at 37°C. For surface staining of AIM markers, cells were incubated in 1 μg/mL human Fc block (BD Biosciences) and fixable viability solution 780 (BD Biosciences) in PBS for 15 min and washed in PBS. An antibody cocktail containing antibodies against CD3 (clone UCHT1, BD Biosciences), CD4 (clone M-T477, BD Biosciences), CD8 (G42-8, BD Biosciences), CXCR5 (J252D4, BD Biosciences), CD38 (HIT2, BD Biosciences), PD-1 (EHI2.1, BD Biosciences), CD69 (clone FN50, BD Biosciences), CD137 (clone 4B4-1, Biolegend), OX40 (clone Ber-ACT35, Biolegend), CCR7 (clone 2-L1-A, BD Biosciences), and CD45RA (clone HI100, BD Biosciences)were added directly to cells and incubated for a further 30 min at 4°C. Following surface staining, cells were washed twice in PBS. All samples were acquired on a BD FACS Symphony and analyzed using FlowJO software v18 (FlowJo, Ashland, USA). The gating strategy for AIM+ T cells is shown in (Methods S3 ). Data were imported into R v4.2 and visualized with the ggplot2 v3.3.6 package.
3
Comprehensive Immunophenotyping of PBMCs
4
Comprehensive Immunophenotyping of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated through a Ficoll step gradient and stained for cell surface markers with the following fluorochrome-conjugated antibodies: CD38 (HIT-2, BD), IgD (IA6-2, BD), IgM (MHM-88, BioLegend), CD10 (HI10a, BD), CD19 (SJ25C1, BD), CD21 (B-ly4, BD), CD27 (0323, eBiosience), CD138 (B-B4, AbD SeroTec), IgG (SouthernBioTech #2043-09), IgA (G20-359, BD), CD3 (SK7, BD), CD28 (CD28.2, BD), FOXP3 (PCH101, eBioscience), CD8 (SK1, BD), CD4 (RPA-T4, eBioscience), γδ-TCR (B1.1, eBioscience), αβ-TCR (IP26, eBioscience), CD45RO (UCHL1, BD), CD45RA (HI100, BD), CD57 (NK-1, BD), CD31 (WM59, eBioscience), CD62L (DREG-56, BD), CD152/CTLA4 (BNI3, BD). Samples were acquired on a FACS-Canto II flow cytometer (BD) or on a Gallios flow cytometer (Beckman Coulter, Miami, FL) and analyzed using FlowJo version 7.6.5 analysis software (Treestar, Ashland, OR).
5
Isolation and Sorting of B Cell Subsets
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B cells were isolated from fresh antecubital venous blood, as previously described [11 (link)]. Briefly, peripheral blood mononuclear cells (PBMC) were isolated from 100 to 120 ml venous blood of untreated MS patients and healthy volunteers using standard density-gradient centrifugation on Ficoll-Paque (Pharmacia Biotech). Magnetic beads (MACS, Miltenyi Biotec) were used according to the manufacturer’s instructions to isolate CD19+ B cells by positive selection, and their purity was confirmed by flow cytometry (routinely > 98% pure). B cells were then washed and resuspended in serum-free X-Vivo 10 medium (Lonza, Walkersville, MD). For experiments with B cell subsets, total B cells were initially sorted from PBMC by CD19+ MACS separation and then stained for CD20+ (2H7), CD27+ (M-T271), IgD (IA6-2), CD24 (ML5), and CD38 (HIT2), all from BD Bioscience. The total B cells were subsequently sorted (using a BD LSRFortessa, BD Bioscience) into transitional (CD20+CD24+CD38+), naive (CD20+CD27−IgD+), or memory (CD20+CD27+IgD−/+) B cell subsets with routine purity confirmation (typically > 93%).
6
Engraftment and Multilineage Reconstitution in NSG Mice
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Eight-week-old NSG mice were sublethally irradiated (300 rad) and transplanted with 1 × 106 unedited and edited CD34+ cells. Donor chimerism was assessed every 4 weeks as the frequency of human cells in total white blood cells by flow cytometry after red blood cell lysis and staining with human CD45 (BD Biosciences). Upon sacrifice, 16 or 20 weeks after transplantation, the chimeric bone marrow was harvested and assayed for level of engraftment and multilineage reconstitution after staining with the following antibodies: CD45 (2D1), CD19 (HIB19), CD33 (P67.6), CD34 (8G12), and CD38 (HIT2) (BD Biosciences) and CD41 (VIPL3) (Invitrogen). For the secondary transplantations, bone marrow cells were enriched for human CD45+ cells by immunomagnetic separation (hCD45 microbeads, Miltenyi Biotec), and the same number of human cells (5 × 106) was injected into each secondary recipient. Secondary recipients were sacrificed 10 weeks after transplantation. All in vivo experiments were conducted with approval from the institutional animal care and use committee.
7
Detailed Immunophenotyping of T-cell Subsets
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The following antibodies were used: CD80 (L307.4; BD Biosciences), CD86 (FUN-1; BD Biosciences), CD86 (IT2.2, BioLegend), CD25 (BC96; eBioscience), FoxP3 (236A/E7; eBioscience), CTLA-4 (BNI3; BD Biosciences), IFN-γ (B27; BD Biosciences), CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD45RA (HI100; eBioscience), CD38 (HIT2; BD Biosciences) and HLA-DR (G46-6; BD Biosciences). For surface marker staining, cells were washed and re-suspended in 50 µl of FACS buffer (PBS and 2% Goat serum) containing antibodies conjugated with fluorochromes. Reactions were incubated for 30 min on ice. Cycling CTLA-4 was stained for 30 min at 37°C. For intracellular staining of FoxP3, a FoxP3 staining kit (eBioscences) was used according to the manufacturer’s instructions. Flow cytometry data were analysed by FlowJo (TreeStar, Ashland, Oregon, USA).
8
Antibodies for Murine and Human Immune Cell Analysis
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Anti–murine antibodies used in this study include AA4.1, CD24 (M1/69), CD21 (7G6), B220 (RA3-6B2), and IgD (11-26C.2A) from BD; BP1 (FG35.4) and CD23 (B3B4) from Invitrogen; IgM (1B4B1), κ (187.1), λ (JC5-1), and SA-HRP conjugated from SouthernBiotech; CD19 (ID3) and IgMa (DS-1) from BioLegend; and Cy5 anti–rabbit polyclonal IgG from Jackson ImmunoResearch Laboratories, Inc. Alexa Fluor 647 anti-M167 (28–6-20) rat IgG2a was provided by S. Porcelli (Albert Einstein College of Medicine, Bronx, NY). Anti–human antibodies used in this study include CD19 (HIB19) and IgM (MHM-88) from BioLegend; CD10 (HI10a), CD24 (ML5), and IgD (IAG-2) from BD; CD27 (323) from eBioscience; and CD38 (HIT2) from BD. Anti–human rat monoclonal, FITC-conjugated 9G4 antibody was provided by I. Sanz.
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