Hematopoietic donor cell chimerism was performed as previously described [15 (link)]. Immune reconstitution was monitored in peripheral blood mononuclear cells (PBMCs) by flow cytometry in between week 0–38. PBMCs were isolated by density gradient centrifugation using Biocoll cell separation solution (Biochrom, Berlin, Germany). Immune cells were stained using the directly fluorescence labeled mAbs CD138-PE (clone: MI15, BD Biosciences, Franklin Lakes, NJ, USA), CD138-PE (clone: MI15, BD Biosciences), CD38-PerCP (clone: HB-7, BD Biosciences), CD19-PacificBlue (clone: SJ25C1, BD Biosciences), CD27-FITC (clone: M-T271, BD Biosciences), IgA-APC (clone: 97924, R&D Sytems, Minneapolis, MN, USA), IgG-APC (clone: 97924, R&D Systems), IgM-APC (clone: IL/41, ThermoFisher), HLA-A2-APC (clone: BB7.2, ThermoFisher), HLA Class I B7-PE (clone: BB7.1, abcam, Cambridge, UK), HLA-Class1 BW6-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) and HLA-Class1 B8-APC (Miltenyi Biotec). Dead cells were excluded from analysis by LIVE/DEAD™ Fixable Aqua Dead Cell Stain (ThermoFisher, Waltham, MA, USA). Measurements were performed using a FACS Canto II or FACS Fortessa (BD Biosciences) and data analyzed using the software FlowJo V10 (FlowJo LCC, Ashland, OR, USA).
Cd19 pacificblue
Manufactured by BD
Sourced in United States
CD19-PacificBlue is a fluorochrome-conjugated antibody that binds to the CD19 cell surface antigen. It is designed for use in flow cytometry applications to identify and quantify B cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd138 pe, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Facs fortessa, by BD (1 mentions)
Biocoll cell separation solution, by Harvard Bioscience (1 mentions)
Cd27 fitc, by BD (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Cd38 percp, by BD (1 mentions)
Cd19 pe, by Thermo Fisher Scientific (1 mentions)
B220 apc, by Thermo Fisher Scientific (1 mentions)
Cd4 bv421, by BioLegend (1 mentions)
Cd19 buv395, by BD (1 mentions)
Lsrfortessa x 20 analyzer, by BD (1 mentions)
Cd23 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd8a percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd43 pe, by BD (1 mentions)
Cd24 apc, by BD (1 mentions)
Tcrβ pe, by BD (1 mentions)
Cd19 bb700, by BD (1 mentions)
B220 pe cy7, by BD (1 mentions)
4 protocols using cd19 pacificblue
1
Immune Reconstitution Monitoring by Flow Cytometry
2
Comprehensive B Cell Immunophenotyping
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Bone marrow of Myce and littermate wild‐type control were harvested into single‐cell suspension with red cell lysis and stained with CD19‐Pacific Blue (1D3, BD, NJ, USA), B220‐PE cy7 (RA3‐6B2, BD), CD24‐APC (M1/69, in house), IgM‐FITC (331.12, in house), CD43‐PE (S7, BD). Spleen red cell lysed single cell suspensions were stained with CD19‐BB700 (1D3, BD), B220‐APC (Thermo Fisher, Waltham, USA), CD23‐PE cy7 (B3B4, Thermo Fisher), CD21‐BV421 (7E9, BioLegend, San Diego, USA), CD138‐PE (281–2, BD). The peritoneal cavity wash was stained with CD19‐PE (ID2, in house), B220‐APC (RA3‐6B2, Thermo Fisher), CD23‐PE cy7 (B3B4, Thermo Fisher), CD5‐BB700 (53–7.3, BD), CD11b‐eFlour450 (M1/70, Thermo Fisher). Lymph node and thymus of single cell suspensions were stained with CD19‐BUV395 (1D3, BD), (TCRβ‐PE (H57‐597, BD), CD4‐BV421 (GK1.5, BioLegend) and CD8a‐PerCp/Cy5.5 (53–6.7, eBioscience, San Diego, USA). Flow cytometry analysis was performed on a BD LSRFortessa X‐20 analyzer (BD).
3
Intracellular Cytokine Staining Workflow
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Intracellular cytokine staining was performed as previously described(1) using the following markers: CD3-APCH7, CD107a PECy7, CD14-Pacific Blue, CD16-Pacific Blue, CD4-PECy5.5, IFN-γ-PerCPCy5.5, CD45RO-AF700, CD19-Pacific Blue (BD Biosciences, San Jose, CA), TNFα-AF647, CD8-BV570, CCR7-BV711 (BioLegend, San Diego, CA), granzyme B-PE Texas Red (Invitrogen), CD27-PECy5 (eBioscience, San Diego, CA) perforin-FITC(Abcam, Cambridge, UK). Prepared cells were acquired using an LSR II flow cytometer equipped with BD FACSDiva software (BD Biosciences). Acquired data was analyzed using the FlowJo software version 7.6.3 (Tree Star).
4
Multiparametric flow cytometry analysis
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Before staining, cells were washed in ice-cold PBS and stained with Live/Dead fixable aqua fluorescent stain (Invitrogen, Carlsbad, CA, USA) for 20 min at 4°C. After washing in FACS buffer (PBS with 0.05% sodium azide and 3% FCS), cells were stained with anti-CXCR5 biotin, anti-GL7 FITC, anti-ICOS PE, anti-PD1 PE-Cy7, anti-CD4 APC-Cy7, and CD19-pacific blue (BD Biosciences) in the presence of Fc receptor blocking antibody (clone 2.4G2, in-house) for a further hour at 4°C, then washed. Streptavidin-APC was added, and samples incubated for 30 mins on ice. Staining for intracellular transcription factors and/or cytokines (Bcl-6 and IFN-γ) were performed following surface staining using fix/perm buffer set (eBioscience, San Diego, CA, USA). Samples were acquired on a LSR II flow cytometer (BD Bioscience, San Diego, CA, USA) using BD FACS-Diva software, and data analyzed using FlowJo software (Tree Star Inc., San Carlos, CA, USA).
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