Cd45r b220 pe cy7

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CD45R/B220-PE-Cy7 is a fluorescently labeled antibody that binds to the CD45R/B220 antigen expressed on the surface of B cells and certain other leukocytes. It can be used for the identification and enumeration of these cell types in flow cytometric analyses.

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CD45R/B220 (19 citations) B220-PE (15 citations) B220 PE-Cy7 (6 citations) CD45R-PE (2 citations)

3 protocols using cd45r b220 pe cy7

Multiparameter Flow Cytometry Immunophenotyping

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Fc-gamma receptors were blocked with a rat anti-mouse CD16/CD32 antibody (BD). Mouse-specific antibodies were the following: CD3-Brilliant violet 510, CD25-PercP Cy5.5, I-A/I-E-Brilliant violet 510, CD45R/B220-PE-Cy7, CD43-APC, CTLA-4-APC (all from BioLegend), CD4-APC-Cy7, CD69-PE, CD11c-PercP Cy5.5, CD19-APC-Cy7, CD40-FITC (all from BD Pharmingen), CD8α-PE-Cy7, FoxP3-FITC, CD86-APC, CD83-FITC, CD80-PE-Cy7, IgM-PercP-eFluor 710 (all from eBioscience).
During the experimental set-up, CD3 antibody was included in the staining panel (BV510 BioLegend clone 17A2) and used for the gating of T cells. The results obtained with gating on CD3+ CD4+ CD8- were similar to those obtained with gating on CD4+ CD8-. Due to the limitation in the maximum number of colours that can be discriminated with the FACS Canto II, CD3 staining was omitted in subsequent stainings.
Dead cells were excluded using the fixable viability dye-eFluor 450 (eBioscience), and intracellular staining was performed using the fixation/ permeabilization buffer set from eBioscience. Flow cytometry measurements were performed on a FACS Canto II instrument (BD) and the data were analyzed with FlowJo software (Tree Star).

Sordé L., Spindeldreher S., Palmer E, & Karle A. (2017). Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?. PLoS ONE, 12(10), e0186046.

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Phenotyping B-cell and T-regulatory subsets

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For analysis of B‐cell populations and T regulatory cells, live single‐cell suspensions were prepared from spleens and stained with propidium iodide (PI; catalog number P4864; Sigma/Merck), CD45R/B220‐PE‐Cy7 (1/400; catalog number RA3‐6B2; BioLegend, San Diego, CA, USA), CD138‐PE (1/400; catalog number 281–2; BioLegend), GL7‐Pacific Blue (1/200; BioLegend), peanut agglutinin (PNA)–fluorescein isothiocyanate (1/400; catalog number L7381; Sigma/Merck), IgG1‐APC (1/400; catalog number RMG1‐1; BioLegend), CD4‐Pacific Blue (1/400; catalog number RM4‐5; BD Biosciences), CD25‐APC (1/100; catalog number PC61; BD Biosciences) and FoxP3‐PE (catalog number MF23; BD Biosciences). Antibodies were diluted in FACS buffer (Hanks' Balanced Salt Solution with 2% fetal calf serum). For staining with anti‐FoxP3, cells were fixed and permeabilized with the Mouse FoxP3 buffer set (BD Biosciences). B‐cell populations were defined as follows: plasma cells, B220^lowCD138^hi; conventional B cells, B220^hi CD138^low; GC B cells, B220^hiGL7^hiPNA^hi; non‐GC B cells, B220^hiGL7^lowPNA^low; isotype switched, B220^hiGL7^hiPNA^hiIgG1^hi; nonisotype switched, B220^hiGL7^hiPNA^hiIgG1^low. T regulatory cells were identified as CD4⁺CD25⁺FoxP3⁺. Cells were analyzed on a BD FACSCanto II (BD Biosciences) after exclusion of PI⁺ dead cells. Data were analyzed using FlowJo version 10.6.2 cell cycle analysis software (BD Biosciences).

Hasnat M.A., Cheang I., Dankers W., Lee J.P., Truong L.M., Pervin M., Jones S.A., Morand E.F., Ooi J.D, & Harris J. (2022). Investigating immunoregulatory effects of myeloid cell autophagy in acute and chronic inflammation. Immunology and Cell Biology, 100(8), 605-623.

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Delivery Efficiency of ASO or Toc-HDO in T Cells

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The delivery efficiency of ASO or Toc-HDO into primary T cells in vivo and EL4 in vitro cells was assayed by flow cytometry using Alexa Flour 647-labeled ASO. Cells were harvested at the indicated time points and washed in PBS by centrifugation at 500 ×;g for 5 min. The cells were then washed and suspended in PBS with 2% BSA and 0.05% sodium azide (Sigma-Aldrich). Surface marker expression levels were determined using the following antibodies: CD3-PE (BioLegend, clone 17A2, 100205), CD45R/B220-PECy7 (BioLegend, clone RA3-6B2, #103221) or CD45R/B220-FITC (BioLegend, clone RA3-6B2, #103205), and CD49d-APC (BioLegend, clone R1-2, #103621). All related antibodies are listed in Supplementary Table 2. Cells were then sorted using a BD FACSAria cell sorter, BD FACSDiva software, and FlowJo v10 software (BD Biosciences) or analyzed using a BD FACSVerse cell analyzer and BD FACSuite software (BD Biosciences). The sorting/gating strategy is shown in Supplementary Fig. 2g.

Ohyagi M., Nagata T., Ihara K., Yoshida-Tanaka K., Nishi R., Miyata H., Abe A., Mabuchi Y., Akazawa C, & Yokota T. (2021). DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo. Nature Communications, 12, 7344.

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