B220 pecy7

Manufactured by Thermo Fisher Scientific

The B220-PECy7 is a laboratory instrument used for flow cytometry applications. It is designed to detect and analyze cells based on specific surface markers labeled with the PECy7 fluorophore. The core function of the B220-PECy7 is to provide accurate and reliable measurements of PECy7-labeled cells within a sample.

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6 protocols using b220 pecy7

Phenotypic Characterization of Immune Cells

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Cells were obtained from peripheral blood (PB), spleen (SPL), bone marrow (BM), peritoneal tumor, and ascites from recipient mice and stained for the combination of the following antibodies: IgM-PE, B220-PECy7, CD138-APC, and CD38-PE (ThermoFisher Scientific). Samples were analyzed with FACS on a Fortesa machine (Becton Dickinson, NJ, U. S. A.) using Cell Quest software (Becton Dickinson). FACS data were analyzed and represented with Flowjo10.

Qiu Q., Li M., Yang L., Tang M., Zheng L., Wang F., Qiu H., Liang C., Li N., Yi D., Yi Y., Pan C., Yang S., Chen L, & Hu Y. (2019). Targeting glutaminase1 and synergizing with clinical drugs achieved more promising antitumor activity on multiple myeloma. Oncotarget, 10(57), 5993-6005.

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Immune Cell Profiling of Murine Kidney

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Right kidney was harvested and immediately processed for flow cytometric analysis. The following antibodies were used: anti CD45-V450, Ly6G APC-Cy7, B220 PE-Cy7, CD3 FITC, CD4 PE, CD8 PerCP-Cy5.5 (ThermoFisher Scientific, Waltham).^{42 (link),43} Details are reported in Suppl. Methods.

Federti E., Matte A., Recchiuti A., Garello F., Ghigo A., El Nemer W., Terreno E., Amoresano A., Mattoscio D., Turrini F., Lebouef C., Janin A., Pantaleo A., Russo R., Marin M., Iatcencko I., Riccardi V., Siciliano A., Iolascon A., Brugnara C, & De Franceschi L. (2023). In Humanized Sickle Cell Mice, Imatinib Protects Against Sickle Cell–Related Injury. HemaSphere, 7(3), e848.

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Clonal Expansion of SLAM Cells

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Serum-free CM was prepared by incubating serum-free medium (BIT; STEMCELL Technologies), 40 μg/ml low-density lipoproteins (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, and 10⁻⁴ M β-mercaptoethanol for 3 days on irradiated (30 Gy) confluent pLKO.1 (pLKO.1-CM) and shCtgf (shCtgf-CM) stromal cells. Before use, CM was filtered through a 0.4 μm filter.
CD34⁻ and CD34⁺ SLAM cells were sorted into round-bottomed 96-well plates preloaded with 100 μl of pLKO.1-CM and shCtgf-CM, supplemented with mSCF (100 ng/ml) and IL-11 (20 ng/ml), both from R&D Systems, and rCTGF (250 ng/ml; BioVendor) where indicated. Immediately after sorting, the plates were centrifuged for 5 min at 200 × g and microscopically inspected for the presence of single cells. Each well was inspected every 24 hr for clonal growth. After 5 days, each clone was harvested and studied for colony formation in growth factor-supplemented methylcellulose (M3434; STEMCELL Technologies), After 10 days, the number of colonies was counted and cells were harvested, and washed three times with HF2⁺, pelleted, and stained with B220-PECy7, CD3ε-PECy5.5, CD11b-APCCy7, GR1-PB, and TER119-PE (eBiosciences). Immunofluorescence staining was measured on a CyAn ADP Lx P8 (Coulter-Cytomation) and analyzed with FlowJo software (TreeStar).

Istvánffy R., Vilne B., Schreck C., Ruf F., Pagel C., Grziwok S., Henkel L., Prazeres da Costa O., Berndt J., Stümpflen V., Götze K.S., Schiemann M., Peschel C., Mewes H.W, & Oostendorp R.A. (2015). Stroma-Derived Connective Tissue Growth Factor Maintains Cell Cycle Progression and Repopulation Activity of Hematopoietic Stem Cells In Vitro. Stem Cell Reports, 5(5), 702-715.

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Isolation of Liver Non-Parenchymal Cells

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Mice were anesthetized, livers perfused with sterile PBS, and non‐parenchymal liver cells isolated as previously described (with slight modifications).24 Briefly, after successful perfusion, whole livers were excised and minced, incubated with 0.2 mg/mL collagenase IV buffer at 37°C for 30 minutes with shaking, double‐passed through 70 μm nylon mesh and centrifuged twice for 3 minutes at 40 g to pellet hepatocytes. Supernatants containing non‐parenchymal cells were purified in the Percoll gradient (Sigma‐Aldrich). Isolated cells were counted in hemocytometer (Bürker‐Türk chamber) and labelled with fluorescent‐conjugated antibodies: CD45‐APC (eBioscience), CD3‐APCeF780 (eBioscience), B220‐PECy7 (eBioscience), NK1.1‐PE (eBioscience), CD11b‐PECy7 (eBioscience), F4/80‐APCeF780 (eBioscience), Ly6G‐PE (BD Pharmingen) and CD95‐AF488 (eBioscience). CD16/CD32 (eBioscience) was used for blocking of Fc receptors, while dead cells were excluded based on 7‐amino‐actinomycin D (7‐AAD; BioLegend) binding. Upon labelling, cells were acquired and analysed using the Attune instrument (Thermo Fisher Scientific) and FlowJo software (FlowJo). The gating strategy is shown in Supporting Information (Figure S1).

Markotic A., Flegar D., Grcevic D., Sucur A., Lalic H., Turcic P., Kovacic N., Lukac N., Pravdic D., Vukojevic K., Cavar I, & Kelava T. (2020). LPS‐induced inflammation desensitizes hepatocytes to Fas‐induced apoptosis through Stat3 activation—The effect can be reversed by ruxolitinib. Journal of Cellular and Molecular Medicine, 24(5), 2981-2992.

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Isolating Murine Splenic B and Plasma Cells

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Mouse spleens were processed into single cell suspensions, pooled in groups of 3–4 and washed before resuspending Ficoll-isolated mononuclear cells in PBS containing 0.5% BSA and antibodies B220-PECy7 (eBioscience, #25-0452-82) and CD138-APC (BD Pharmigen #558626). After one wash, cells were resuspended at 20 million/mL and DAPI (1 μg/mL) added to exclude dead cells. Cells collected were B220+ CD138- (B cells), and CD138+ (plasma cells), and were >95% pure. Bone marrow extracted from mouse femurs and spleen mononuclear cells were also analyzed by FACS with B220, CD138, CD3, CD11b and Gr1 antibodies.

Wei X., Calvo-Vidal M.N., Chen S., Wu G., Revuelta M.V., Sun J., Zhang J., Walsh M.F., Nichols K.E., Joseph V., Snyder C., Vachon C.M., McKay J.D., Wang S.P., Jayabalan D.S., Jacobs L.M., Becirovic D., Waller R.G., Artomov M., Viale A., Patel J., Phillip J., Chen-Kiang S., Curtin K., Salama M., Atanackovic D., Niesvizky R., Landgren O., Slager S.L., Godley L.A., Churpek J., Garber J.E., Anderson K.C., Daly M.J., Roeder R.G., Dumontet C., Lynch H.T., Mullighan C.G., Camp N.J., Offit K., Klein R.J., Yu H., Cerchietti L, & Lipkin S.M. (2018). Germline Lysine-Specific Demethylase 1 (LSD1/KDM1A) Mutations Confer Susceptibility to Multiple Myeloma. Cancer research, 78(10), 2747-2759.

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Hematopoietic Stem Cell Transplantation

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Cell preparations were injected intravenously into irradiated (9.5 Gy) recipients that differed in their CD45 isoform expression. For the primary transplants, E11 AGM, E12 Plac, E12 FL, and E14 FL were injected at 1, 0.5, 0.5, and 0.004 embryo equivalent per recipient, respectively, together with 100,000 total BM helper cells. For the secondary and tertiary transplants, between two and three million total BM cells (without helper cells) were injected, which were normalized according to the level of donor chimerism. At the indicated time points, donor contribution to the peripheral blood was analyzed by flow cytometry using CD45.1-PE (eBioscience A20), CD45.2-FITC (eBioscience 104), CD11b-eFluor450 (eBioscience M1/70), Gr1-AF700 (BioLegend RB6-8C5), CD19-BV605 (BioLegend 1D2), B220-PECy7 (eBioscience RA3-6B2), CD3-APC (BioLegend I45-2C11), and ckit-APCeFluor780 (eBioscience 2B8). Mice were considered positive for repopulation if the donor contribution to the total peripheral blood was >5%. Peripheral blood counts were performed on an ABC blood counter (Woodley).

Barrett N.A., Malouf C., Kapeni C., Bacon W.A., Giotopoulos G., Jacobsen S.E., Huntly B.J, & Ottersbach K. (2016). Mll-AF4 Confers Enhanced Self-Renewal and Lymphoid Potential during a Restricted Window in Development. Cell Reports, 16(4), 1039-1054.

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