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B220 apc cy7

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The B220-APC-Cy7 is a fluorescently labeled antibody product designed for flow cytometry applications. It targets the B220 antigen, also known as CD45R, which is expressed on the surface of B cells and certain other leukocytes. The APC-Cy7 fluorophore combination provides a specific emission spectrum for detection and analysis.

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6 protocols using b220 apc cy7

1

Comprehensive Immune Cell Profiling

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Peripheral blood was collected by facial vein bleed into EDTA Microtainer tubes (BD Biosciences, NJ) and CBC performed on a Heska Hematrue (Heska, CO). Flow cytometry was acquired on a FACSVerse (BD Biosciences, NJ) and analyzed using Flowjo (Flowjo, OR). Cell sorting was performed on an Astrios (Beckman Coulter). Antibodies were used from (1) BioXCell: Fc block (2.4G2), and (2) eBioscience: B220-APC-Cy7 (RA3–6B2), CD11c-APC (N418), CD138-PE-Cy7 (DL-101), CD19-APC (1D3), CD19-PE-Cy7 (1D3), CD21-FITC (4E3), CD23-PE (B3B4), CD25-APC (PC61.5), CD3-FITC (145–2C11), CD4-PE-Cy7 (RM4–5), CD43-APC (R2/60), CD44-PE (IM7), CD45.1-PE (A20), CD45.2-APC (104), CD5-APC (53–7.3), CD62L-APC (MEL-14), CD8-APC-Cy7 (53–6.7), CD8-FITC (53–6.7), Foxp3-PE (FJK-16s), IFNγ-APC (XMG1.2), IgD-FITC (11–26c), IgM-PE (eB121–15F9), IL-17-APC (eBio1787), NK1.1-PB (PK136). Cell viability assessed using fixable Live/Dead Aqua dye (Invitrogen). Flow cytometry for LC3 was performed per manufacturer’s instructions (FlowCellect, Millipore).
Khor B., Conway K.L., Omar A.S., Biton M., Haber A.L., Rogel N., Baxt L.A., Begun J., Kuballa P., Gagnon J.D., Lassen K.G., Regev A, & Xavier R.J. (2019). Distinct tissue-specific roles for the disease-associated autophagy genes ATG16L2 and ATG16L1. Journal of immunology (Baltimore, Md. : 1950), 203(7), 1820-1829.
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2

Single Cell Isolation and Immunophenotyping

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In order to obtain single cell suspensions, spleen and mesenteric lymph node (MLN) tissues were passed through 70μm filters in a PBS solution supplemented with 3% fetal bovine serum. Bone marrow (BM) was flushed from the long bones of mice with 22 ½ gauge syringes using 3% FBS in PBS, and then subsequently passed through 70μm filters. Red blood cells were then removed from the single cell suspensions by lysis with Ammonium-Chloride-Potassium buffer.
An equal number of cells for each sample were first blocked with FcγR for 20 minutes, followed by one wash with PBS and incubation with the primary antibody (1:100–1:500) for 30 minutes at room temperature in the dark. When secondary antibodies were necessary, samples were incubated with the conjugated antibody for 30 minutes after primary antibody incubation. Samples were then washed once with PBS, transferred to FACS tubes, and passed through either BD LSR II flow cytometer or Fortessa FACS machines for analysis. Data were analyzed using FlowJo software v8.8.2 (Tree Star, Ashland, OR). Antibodies were purchased from eBioscience (San Diego, CA): c-kit APC #17-1171-82, Stem cell antigen-1 (Sca) PE-Cy7 #25-5981-82, Myeloid differentiation antigen (Gr1) APC-Cy7 #47-5931-82, Macrophage-1 antigen (Mac1) APC-Cy7 #47-0112-82, B220 APC-Cy7 #47-0452-82, CD4 APC-Cy7 #47-0042-82, CD8 APC-Cy7 #47-0081-82.
Danielson L.S., Reavie L., Coussens M., Davalos-Vega V., Castillo-Martin M., Guijarro M., Coffre M., Cordon-Cardo C., Aifantis I., Ibrahim S., Liu C., Koralov S, & Hernando E. (2014). Limited miR-17-92 overexpression drives hematologic malignancies. Leukemia research, 39(3), 335-341.
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3

Multi-lineage Hematopoietic Cell Analysis

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Total white blood cells from hematopoietic organs were stained in PBS supplemented with 2% FBS with fluorochrome-conjugated mouse antibodies against specific hematopoietic lineage markers. For the analysis of transplant-receiving mice, WBM was stained with fluorochrome-conjugated mouse antibodies to discriminate cells derived from competitors and donors (CD45.1+ and CD45.2+ respectively) and GFP expression was used to precisely define donor-derived cells (GFP+ CD45.2+). Fluorochrome-conjugated mouse antibodies were obtained from Becton Dickinson (Streptavidin/PeCy5, CD117/PeCy7, CD45.1/PE, CD4/PE or PB, CD8/PeCy7 or APC, CD19/APC, B220/APCCy7, TCRβ/PE, CD279/BV421, CD11b/PerCP-Cy5.5, Annexin V/APC, Ki67/PE) and eBiosciences (CD117/APCCy7, Sca-1/APC, CD45.2/APC, Gr1/PE). Hoescht 33342 was obtained from Invitrogen. APC BrdU Flow kit (Becton Dickinson) was used according to the manufacturer’s instruction. Cell sorting was performed either on a MoFlow (Beckman Coulter) or Influx (Becton Dickinson) cell sorter and analysis on a Canto II (Becton Dickinson). FACS data were analyzed by FlowJo Software (v8.8.7).
Scourzic L., Couronné L., Pedersen M.T., Della Valle V., Diop M., Mylonas E., Calvo J., Mouly E., Lopez C.K., Martin N., Fontenay M., Bender A., Guibert S., Dubreuil P., Dessen P., Droin N., Pflumio F., Weber M., Gaulard P., Helin K., Mercher T, & Bernard O.A. (2016). DNMT3AR882H mutant and Tet2 inactivation cooperate in the deregulation of DNA methylation control to induce lymphoid malignancies in mice. Leukemia, 30(6), 1388-1398.
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4

Multi-lineage Hematopoietic Cell Analysis

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Total white blood cells from hematopoietic organs were stained in PBS supplemented with 2% FBS with fluorochrome-conjugated mouse antibodies against specific hematopoietic lineage markers. For the analysis of transplant-receiving mice, WBM was stained with fluorochrome-conjugated mouse antibodies to discriminate cells derived from competitors and donors (CD45.1+ and CD45.2+ respectively) and GFP expression was used to precisely define donor-derived cells (GFP+ CD45.2+). Fluorochrome-conjugated mouse antibodies were obtained from Becton Dickinson (Streptavidin/PeCy5, CD117/PeCy7, CD45.1/PE, CD4/PE or PB, CD8/PeCy7 or APC, CD19/APC, B220/APCCy7, TCRβ/PE, CD279/BV421, CD11b/PerCP-Cy5.5, Annexin V/APC, Ki67/PE) and eBiosciences (CD117/APCCy7, Sca-1/APC, CD45.2/APC, Gr1/PE). Hoescht 33342 was obtained from Invitrogen. APC BrdU Flow kit (Becton Dickinson) was used according to the manufacturer’s instruction. Cell sorting was performed either on a MoFlow (Beckman Coulter) or Influx (Becton Dickinson) cell sorter and analysis on a Canto II (Becton Dickinson). FACS data were analyzed by FlowJo Software (v8.8.7).
Scourzic L., Couronné L., Pedersen M.T., Della Valle V., Diop M., Mylonas E., Calvo J., Mouly E., Lopez C.K., Martin N., Fontenay M., Bender A., Guibert S., Dubreuil P., Dessen P., Droin N., Pflumio F., Weber M., Gaulard P., Helin K., Mercher T, & Bernard O.A. (2016). DNMT3AR882H mutant and Tet2 inactivation cooperate in the deregulation of DNA methylation control to induce lymphoid malignancies in mice. Leukemia, 30(6), 1388-1398.
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5

Evaluating Peritoneal Myeloid Cells

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Either 200ul PBS (control) or 20ug of recombinant MCSF (rhMCSF, Novartis) was injected into the peritoneums (i.p.) of wild type, C57/B6J 8–10 week old mice at indicated times prior analysis. For cell cycle analysis, mice were also injected i.p. with 2 mg of BrdU (BD Pharmingen) dissolved in PBS, as per manufacturers instructions 4h prior to analysis. To harvest peritoneal cells mice were sacrificed and subsequently injected with 10mL ice-cold PBS containing 2mM EDTA (Sigma). Intra-peritoneal washouts were collected in 50mL Falcon tubes. Red cell lysis was performed on total washouts (RBC Lysis Buffer, Invitrogen) prior to staining for cell surface markers and cell cycle analysis or FACS sorting. Remaining mononuclear cells were then stained using the following antibody cocktail in the presence of FcBlock (BD Biosciences): CD11b-PE-Cy7 (BD Biosciences), MHC-II-FITC, B220-APC-Cy7, F4/80-PE (all from eBioscience) and Fixable Aqua Dead-V500 (Life Technologies) and either sorted for single cell analysis or further processed for cell cycle analysis.
Soucie E.L., Weng Z., Geirsdóttir L., Molawi K., Maurizio J., Fenouil R., Mossadegh-Keller N., Gimenez G., VanHille L., Beniazza M., Favret J., Berruyer C., Perrin P., Hacohen N., Andrau J.C., Ferrier P., Dubreuil P., Sidow A, & Sieweke M.H. (2016). Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells. Science (New York, N.Y.), 351(6274), aad5510.
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6

Multicolor Flow Cytometry Antibody Panel

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Commercial antibodies and staining reagents, from BioLegend: GL7-PacBlue, GL7-PE, GL7-A647, B220-PerCP-Cy5.5, B220-APC-Cy7, IgMb-FITC, IgMa-PE, CD45.2-FITC, CD45.2-APC, CD45.1-FITC, CD45.1-PE, IgD-PB, CD21/35 (7E9)-PE, CD138-PE, CD38-PE-Cy7, CD31-A647, CD157-PE, Streptavidin-PE/Cy7; from eBioscience: CD95 (APO-1/Fas)-PE (clone 15A7) and viability dye Fixable live/dead stain Efluor780; from ThermoFisher Scientific: Rabbit-anti-Goat-A488, Hoechst 33342, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), Phalloidin-A568; from Southern Biotech: AP-Goat-anti-Mouse IgG, AP-Goat-anti-Mouse IgG2a, AP-Goat-anti-Mouse IgG2c; from Rockland Immunochemicals: Rabbit polyclonal anti-B-Phycoerythrin; from DAKO: Rabbit polyconal anti-Mouse Immunoglobulins-biotin; from Perkin-Elmer: Europium-labeled streptavidin. In-house generated anti-idiotypic Ab, clone 9D11, conjugated to Alexa Fluor 647 or Alexa Fluor 568; in-house generated Rabbit polyclonal anti-C3b conjugated to Alexa Fluor 633.
Degn S.E., van der Poel C.E., Firl D.J., Ayoglu B., Al Qureshah F.A., Bajic G., Mesin L., Reynaud C.A., Weill J.C., Utz P.J., Victora G.D, & Carroll M.C. (2017). Clonal Evolution of Autoreactive Germinal Centers. Cell, 170(5), 913-926.e19.
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