Cells from the bone marrow, thymus, spleen, liver, and peripheral blood were stained and analyzed on a FACSCanto (BD Biosciences) running FACSDiva (v.6.1.3) and FlowJo (v.7.6.1, FlowJo LLC) software. Lymphocytes were identified and gated based on their forward-scattered light (FSC) and side-scattered light (SSC) profiles. Cell suspensions were treated with ACK lysis buffer (150 mmol/L NH4Cl, 10 mmol/L KHCO3, and 0.1 mmol/L disodium EDTA) and then stained with the following conjugated antibodies: B220-APC-Cy7 (BD Biosciences), CD2-FITC (BD Biosciences), CD3ε-PE (BD Biosciences), CD3ε-Alexaflore 647 (BD Biosciences), CD4-FITC (Caltag Laboratories), CD8a-APC (Caltag Laboratories), CD8a-Alexaflore 647 (BD Biosciences), CD25-FITC (BD Biosciences), CD44-PE (BD Biosciences), CD45-APC (BD Biosciences), Ki-67-FITC (BD Biosciences), Ly5.1-PerCP-Cy5.5 (BD Biosciences), Ly5.2-PerCP-Cy5.5 (BD Biosciences), NK1.1-APC (Caltag Laboratories), and Sca1-Alexaflore 647 (BD Biosciences).
B220 apc cy7
Manufactured by BD
Sourced in Australia, United States
The B220-APC-Cy7 is a fluorescently labeled antibody designed for flow cytometry applications. It targets the B220 antigen, which is expressed on the surface of B cells. The antibody is conjugated with Allophycocyanin (APC) and Cyanine 7 (Cy7) dyes, which can be detected using standard flow cytometry equipment.
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Lab products found in correlation
Cd45.2 pe, by BD (3 mentions)
Cd11b pe cy7, by BD (3 mentions)
Cd3 fitc, by BD (3 mentions)
Gr1 fitc, by BD (2 mentions)
Cd4 pe cy7, by BD (2 mentions)
Cd45.1 apc, by BD (2 mentions)
Ki67 fitc, by BD (1 mentions)
Cd2 fitc, by BD (1 mentions)
Cd3ε pe, by BD (1 mentions)
Cd25 fitc, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Cd45 apc, by BD (1 mentions)
Cd44 pe, by BD (1 mentions)
Gallios cytometer, by Beckman Coulter (1 mentions)
7 aminoactinomycin d, by Thermo Fisher Scientific (1 mentions)
Fixable viability stain 510, by BD (1 mentions)
Facscan, by BD (1 mentions)
Cd3 pe cy7, by BD (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
9 protocols using b220 apc cy7
1
Multiparameter Flow Cytometry Analysis
2
Multiparameter Flow Cytometry Analysis
3
Multiparameter Flow Cytometry Analysis
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RPMI 1640, FBS, penicillin, and streptomycin were obtained from Invitrogen (Grand Island, NY). The liver dissociation kit, recombinant murine was from Miltenyi Biotec. Phorbol 12-myristate 13-acetate (PMA), brefeldin A, ionomycin, CD3, CD28, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). We obtained the following fluorescein-conjugated anti-mouse antibodies from eBioscience (San Diego, CA), Biolegend (San Diego, CA) and BD: CD3e-APC-Cy7 (145-2C11), CD4-PerCP-Cy5.5 (RM4-5), CD8a-PE (53-6.7), ICOS-PE-Cy7 (C398.4A), CD69-BV421 (MIH5), CD19- PE-Cy5(6D5), CD138-PE-Cy7 (281-2), B220-APC-Cy7 (RA3632), CD80-PE (16-10A1), CD86-APC (GL1), CD3e-FITC (145-2C1), CD11b- PE-Cy7 (M1/70), Ly-6C-PerCP-Cy5.5 (HK1.4), F4/80- APC-Cy7 (3M8), CD192/CCR2-BV421 (SA203G11), CX3CR1-APC (SA011F11), CD135-BV421 (A2F10.1), CD11c- PerCP-Cy5.5 (HL3), Gr-1-FITC (RB6-8C5), Ly-6G- APC-Cy7 (1A8), CD287/TLR7-PE (A94B10), CD103-PE (M290), IFN-γ-APC (XMG1.2), IL-4-PE (11B11), IL-2-PE (JES6-5H4), IL-6-APC (MP5-20F3), IL-10-PE (JES5-16E3), IL-13-eFlour450 (ebio13A), IL-17-PE (TC11-18H10.1)), IL-21-APC (FFA21) and their corresponding isotype controls.
4
Cas9-Induced Immune Response Analysis
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For the analysis of the Cas9-induced immune response, 4-week-old Ntv-a; LSL-Cas9; hUBC-CreERT2 mice were fed ad libitum with tamoxifen containing diet for the duration of the experiment (Supplementary Fig. 3a ).
Complete blood counts were carried out using the Abacus Junior Vet (Diatron). Cells were isolated from spleen and brain by mechanical disruption. Red blood cells were lysed using the red blood cell lysis buffer (Sigma-Aldrich). All cells were stained with CD45-PerCP (Biolegend, #103130, 1:200), CD3-AF700 (eBiosciences, #56-0032, 1:100), CD4-PECy7 (BD Pharmingen, #552775, 1:200), Gr-1-PE (BD Pharmingen, #553128, 1:200), CD11b-PerCPCy5.5 (BD Pharmingen, #550993, 1:30) and B-220-APC-CY7 (BD Pharmingen, #552094, 1:200). Samples were acquired in an LSR Fortessa (BD, San Jose, CA) equipped with 355, 488, 561 and 640 nm lines. We used pulse processing to exclude cell aggregates and DAPI to exclude dead cells. All data were analyzed using FlowJo 9.9.4 (Treestar, Oregon).
Complete blood counts were carried out using the Abacus Junior Vet (Diatron). Cells were isolated from spleen and brain by mechanical disruption. Red blood cells were lysed using the red blood cell lysis buffer (Sigma-Aldrich). All cells were stained with CD45-PerCP (Biolegend, #103130, 1:200), CD3-AF700 (eBiosciences, #56-0032, 1:100), CD4-PECy7 (BD Pharmingen, #552775, 1:200), Gr-1-PE (BD Pharmingen, #553128, 1:200), CD11b-PerCPCy5.5 (BD Pharmingen, #550993, 1:30) and B-220-APC-CY7 (BD Pharmingen, #552094, 1:200). Samples were acquired in an LSR Fortessa (BD, San Jose, CA) equipped with 355, 488, 561 and 640 nm lines. We used pulse processing to exclude cell aggregates and DAPI to exclude dead cells. All data were analyzed using FlowJo 9.9.4 (Treestar, Oregon).
5
Flow Cytometry Analysis of Immune Cell Populations
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Flow cytometry was performed using a FACSCanto 6-colour flow cytometer (BD Biosciences, San Jose CA). The following antibodies were purchased: Sca-1-PE-Cy7, c-Kit-APC, CD43-APC, IgM-PCP.Cy5.5, IgM-Biotin (Australian Biosearch, WangarraWA, Australia); B220-APC.Cy7, B220 PE-Cy5, CD11b-PE, CD11b-FITC, CD19-PE, CD19-APC.Cy7, Gr1-FITC, Streptavidin APC and Lineage Cocktail of biotinylated CD3, Gr-1, Ter119, B220 and CD11b (BD Biosciences, San Jose CA), and Streptavidin Pacific Blue (Thermofisher Scientific, North Ryde, NSW, Australia). Cells were labelled with antibodies as previously described [30 (link)].
6
Evaluating Engraftment of Gene-Modified Cells
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At 7-and 17-week post-transplant, peripheral blood was collected from study animals and analyzed by flow cytometry (FACS) and qPCR to evaluate engraftment of gene-modified cells. Blood was collected from the lateral tail vein in an EDTA-containing tube. RBC Lysis was carried out using an ammonium chloride-based method (Pharmlyse, BO), and the cells were divided for qPCR or FACS analysis. Cells for FACS were stained with antibodies against CD45.1 and CD45.2 to distinguish between host and donor cells and against CD 11b, CD3 and B220 for immunophenotypic characterization (Antibodies used: CD45.1-APC, CD45.2-PE, CD 11b-PECy7, CD3-FITC and B220-APC-Cy7; all from BD Biosciences, San Jose, CA; cat.no. 561873, 560695, 552850, 555274, and 552094, respectively).
7
Comprehensive Immunophenotyping of Murine Cells
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Single cell suspensions from thymus (if large enough), spleen, and bone marrow (from sacrificed animals) were prepared and cells were counted using a hemacytometer or automated cell counter. A subset of cells (approximately 1 x 10 6 ) was used for flow cytometric analyses. In addition, peripheral blood samples were also analyzed by Flow Cytometry. Surface markers used for immunophenotyping included CD45.1, CD45.2, CD 11b, CD8, CD3, CD4, B220 and Gr-I (Antibodies used: CD45.1-APC, CD45.2-PE, CD 11b-PECy7, CD3-FITC and B220-APC-Cy7, CD4-PE-Cy7, CD8a-APC-Cy7, and Gr-1-APC-cy7all from BD Biosciences, San Jose, CA; cat.no. 561873, 560695, 552850, 555274, 552094, 563933, 557654, and 560600, respectively).
8
Multicolor Flow Cytometry of Immune Cells
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The following antibodies were used for staining: anti-mouse IgM-APC, B220-FITC, CD93-PE CF594, CD19-Alexa Fluor 647, IL10-PE, CD23-BB515, CD5-PerCP, CD1d-BV421, CD43-PE Cy7, B220-APC Cy7, Fc Blocker (CD16/CD32), Cytofix/CytoPerm kit (all from BD Pharmingen), CD23-PerCP/Cy5.5, CD93-PE Cy7, CD1d-PB, CD5-PE Cy7, CD21-APC Cy7, CD19-PE, CD43-APC Cy7 (all from Bio Legend), Live/Dead aqua marker (Molecular Probes).
9
Multiparameter Flow Cytometry of Lymphocytes
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Lymphocytes from local lymph nodes were collected and stained with different fluorescence-labeled antibody cocktails as we previously described [27 (link)] to detect the percentages of CD4+ T cells (CD3-FITC, CD4-PE), CD8+ T cells (CD3-FITC, CD8a-APC), IFN-γ secreting cells (CD3-FITC, CD8-APC, IFN-γ-PE-Cy5-5), Tfh (T follicular helper) cells (CD45-APC-Cy7 (BD Pharmingen, San Diego, CA, USA), CD4-FITC (BD Pharmingen, USA), CD185-APC, PD-1-Pacific Blue (BD Pharmingen, USA)), germinal center (GC) B cells (B220-APC-Cy7, CD45-APC-Cy7 (BD Pharmingen, USA), CD95-PE, GL-7-APC), and plasma cells (B220-Pacific Blue, CD27-PE-Cy7, CD138-PE). Except where indicated, the remaining antibodies were purchased from BioLegend, San Diego, CA, USA. The stained cells were assessed by flow cytometry (BD LSRFortessa TM Cell Analyzer, San Jose, CA, USA), followed by FlowJo software analysis.
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