PB was collected in heparin-coated tubes (Venosafe plastic tubes, Terumo Europe N.V., Leuven, Belgium) and PB mononuclear cells (PBMC) were isolated using high density centrifugation (Lympholyte; Cedarlane Laboratories, SanBio B.V., Uden, the Netherlands). PBMC (0.5×106 cells) were stained using anti-human CD19 PerCP-Cy5.5 and CD4 APC to discriminate between B and T cells, respectively (BD Biosciences, Erembodegem, Belgium). B cell subpopulations and surface molecules were defined using following anti-human antibodies: IgD APC-Cy7, CD27 PE-Cy7, HLA-DR/DP/DQ (major histocompatibility complex (MHC)-II) FITC, CD80 PE and CD86 PE-CF594 (all from BD Biosciences, Erembodegem, Belgium). Following anti-human monoclonal antibodies were used for T cell analysis: CD45RA APC-H7, CD45RO PE-CF594, CXCR5 Alexa Fluor 488 and PD-1 PE-Cy7 (all from BD Biosciences, Erembodegem, Belgium), CD25 PerCP-Cy5.5 and CD127 PE (eBioscience, San Diego, USA). Following isotype controls were used: mouse IgG1 PErCP-Cy5.5, IgG1 PE, IgG1 PE-Cy7, IgG2aκ PE-CF594, IgG2bκ APC-H7, IgG1 APC, IgG2aκ FITC, IgG1κ PE-CF594, IgG1 PE-Cy7, IgG2aκ APC-H7 and rat IgG2b Alexa Fluor 488 (all from BD Biosciences, Erembodegem, Belgium). All flow cytometric analyses were performed on a FACSAriaII flow cytometer and analyzed with FACS Diva software (BD Biosciences).
Igg1 pe cy7
Manufactured by BD
Sourced in Belgium
IgG1-PE-Cy7 is a fluorochrome-conjugated antibody that binds to the IgG1 isotype of immunoglobulins. It is used in flow cytometry and other immunoassays to detect and quantify IgG1-expressing cells or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Igg1 apc, by BD (3 mentions)
Facsdiva software, by BD (2 mentions)
Igg1 pe, by BD (2 mentions)
Cd73 pe cy7, by BD (1 mentions)
Cd90 bv421, by BD (1 mentions)
Cd146 pecy7, by BD (1 mentions)
Cd34 pe, by Miltenyi Biotec (1 mentions)
Ng2 pe, by R&D Systems (1 mentions)
Facsdiva 6, by BD (1 mentions)
Cd105 pe, by BD (1 mentions)
Cd19 pe, by BD (1 mentions)
Hla dr pe, by BD (1 mentions)
Cd44 apc, by Miltenyi Biotec (1 mentions)
Cd45 apc, by BD (1 mentions)
Cd11b pe, by BD (1 mentions)
Aria 3, by BD (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Facs tube, by BD (1 mentions)
Lsrii 4 laser flow cytometer, by BD (1 mentions)
Ammonium chloride, by Merck Group (1 mentions)
5 protocols using igg1 pe cy7
1
Comprehensive B and T Cell Immunophenotyping
2
Flow Cytometry Immunophenotyping of Cells
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Flow cytometry analysis for hematopoietic and non-hematopoietic cell surface markers was performed on freshly isolated mononuclear cells and on adherent cell populations at p0 and p1. Cells were incubated with single or combinations of the following mouse monoclonal anti-human antibodies in PBS containing 1% fetal calf serum for 30 min at 4 °C: CD45-Alexa 488 (1:20, Beckman Coulter, Nyon, Switzerland), CD45-APC (1:5), CD73-PE-Cy7 (1:40), CD105-PE (1:40), CD90-BV421 (1:13), CD146-PeCy7 (1:20), HLA-DR-PE (1:5), CD19-PE (1:5), CD11b-PE (1:5, all BD Bioscience), CD44-APC (1:10), CD34-PE (1:10, both Miltenyi Biotec, Bergisch-Gladbach, Germany), NG2-PE (1:10), PDGF-rβ-Alexa 700 (1:20, both R&D Systems). Unstained isotype (IgG1-PeCy7 (1:20, BD Bioscience)) and single antibody controls were included. Flow cytometry was performed using a BD Aria III, and a minimum of 25,000 events were acquired for each sample, and data were analyzed using BD FACS Diva 6.1.3. Appropriate compensation settings and gating were applied in order to account for cellular debris, cell doublets, spectral overlap, and autofluorescence.
3
MSC Enumeration by Flow Cytometry
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MSC enumeration by flow cytometry was performed on the same cohort of donors in order to directly compare the data with the results of the CFU-F assay. For this, 100 μl of BMA was placed in a FACS tube (Falcon) containing 20 μl CD271-allophycocyanine (APC) (Miltenyi Biotec) and 20 μl CD45-phycoerythrin- (PE-) cyanine dye 7 (Cy7) (BD Pharmingen, Oxford, UK) for 15 minutes at room temperature (RT); 10 μl of 7-Aminoactinomycin-D (7-AAD, BD Pharmingen) was added simultaneously in order to distinguish between live and dead cells. After staining, erythroid lineage cells were lysed using ammonium chloride (Sigma, Dorset, UK) at 37°C for 10 minutes. Finally, 50 μl of CountBright absolute counting beads (Life Technologies) was added at RT after a quick vortex for 5-7 seconds, as previously described [27 (link)]. The data were acquired using the Becton Dickinson (BD) LSRII 4 laser flow cytometer. Unstained and single antibody-stained controls were used to optimize the cytometer voltage settings and spectral compensation, and isotype controls (IgG1-APC and IgG1-PE-Cy7, BD Pharmingen) were used to gate for MSCs. The analysis of the flow cytometry data was performed using the FACS Diva software (BD Biosciences, Oxford, UK), as previously described [27 (link)].
4
Multicolor Flow Cytometry of PBMC
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Cryopreserved PBMC were thawed and processed if they had at least 85% viability. They were assessed by 6-color flow cytometry on the same day (FACSCanto, BD Biosciences, Franklin Lakes, USA) after cell staining and they were analyzed using the FlowJo software (TreeStar, San Jose, USA).
The following monoclonal antibodies were used to assess T, B and NK cells: CD3-FITC, CD4-PerCP, CD10-PE-Cy7, CD19-PerCP, CD38-FITC, CD38-PE-Cy7 (BD Biosciences, San Jose, USA); CD3-APC-Cy7, CD4-FITC, CD16-PE-Cy7, CD21-APC, CD27-PE, CD45-APC-Cy7, CD56-PE-Cy7, HLA-DR-PerCP, PD-1-PE (BD, San Jose, USA); 2B4-APC and FcRL4-APC (BioLegend, San Diego, USA). Isotypic controls (IgG1-FITC, IgG1-PE, IgG2a-PerCP, IgG1-PE-Cy7, IgG1-APC, IgG2b-APC, from BD Biosciences, San Jose, USA; IgG1-PE and IgG1-APC, from BioLegend, San Diego, USA) were used to evaluate nonspecific staining.
CD4+T cells were defined as CD3+CD4+ and CD8+T cells were defined as CD3+CD4-, and evaluated for frequencies of activated (CD38+HLA-DR+); or exhausted (PD-1+ or 2B4+) cell subset. B cells were defined as CD3-CD19+, and evaluated for frequencies of transitional (CD10+), naïve (CD10-CD21+CD27-), resting memory (CD10-CD21+CD27+), activated mature (CD10-CD21-CD27+) and exhausted memory B cells (CD10-CD21-CD27- or PD-1+ or FcRL4+).
NK cells were defined as CD45+CD3-CD16+CD56+ and evaluated for frequencies of PD-1+ exhausted cells.
The following monoclonal antibodies were used to assess T, B and NK cells: CD3-FITC, CD4-PerCP, CD10-PE-Cy7, CD19-PerCP, CD38-FITC, CD38-PE-Cy7 (BD Biosciences, San Jose, USA); CD3-APC-Cy7, CD4-FITC, CD16-PE-Cy7, CD21-APC, CD27-PE, CD45-APC-Cy7, CD56-PE-Cy7, HLA-DR-PerCP, PD-1-PE (BD, San Jose, USA); 2B4-APC and FcRL4-APC (BioLegend, San Diego, USA). Isotypic controls (IgG1-FITC, IgG1-PE, IgG2a-PerCP, IgG1-PE-Cy7, IgG1-APC, IgG2b-APC, from BD Biosciences, San Jose, USA; IgG1-PE and IgG1-APC, from BioLegend, San Diego, USA) were used to evaluate nonspecific staining.
CD4+T cells were defined as CD3+CD4+ and CD8+T cells were defined as CD3+CD4-, and evaluated for frequencies of activated (CD38+HLA-DR+); or exhausted (PD-1+ or 2B4+) cell subset. B cells were defined as CD3-CD19+, and evaluated for frequencies of transitional (CD10+), naïve (CD10-CD21+CD27-), resting memory (CD10-CD21+CD27+), activated mature (CD10-CD21-CD27+) and exhausted memory B cells (CD10-CD21-CD27- or PD-1+ or FcRL4+).
NK cells were defined as CD45+CD3-CD16+CD56+ and evaluated for frequencies of PD-1+ exhausted cells.
5
Multicolor Flow Cytometry of Murine Hematopoietic Cells
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Single-cell suspension of bone marrow was obtained by mechanical dissociation using mortar and pestle, and whole spleens were mashed into suspension using a 70-µm cell strainer (BD Falcon). Bone marrow cells, splenocytes, and peripheral blood from mice were red cell-lysed in ACK lysis buffer, and cells were incubated with cell surface FACS antibodies (in FACS buffer; 2% FCS in PBS) for at least 1 h on ice before analysis on a BD LSR II or BD LSR Fortessa flow cytometer. For staining with antibody cocktails, compensation was set using single-stained controls. B220-APCCy7 (103223), B220-APC (103211), and Flt3-PE (135306) were purchased from Biolegend. CD3-PE (561799), Mac1-PerCPCy5.5 (550993), IgD-PE (558597), IgM-APC (550676), CD19-PECy7 (552854), CD24-PerCPCy5.5 (562360), and CD43-PECy7 (562866) were purchased from BD Biosciences. ckit-PerCP eFluor710 (46-1171-80), hTSLPR (CRLF2)-PE (12-5499-42), and hCD127-PECy7 (25-1278-41) were purchased from eBioscience. mTSLPR (Crlf2)-PE (FAB5461P) was purchased from R&D Systems. An IgG isotype antibody was used as a negative control for IL7R (IgG1-PECy7; BD Biosciences, 557872) and CRLF2 (IgG2a-PE; eBioscience, 12-4724-82).
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