Anti b220 pecy7

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States, Germany

The Anti-B220 PeCy7 is a fluorescently-labeled antibody used for the detection and analysis of B220-positive cells in flow cytometry applications. It binds specifically to the B220 antigen expressed on the surface of B cells.

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4 protocols using anti b220 pecy7

Multi-parameter Analysis of Germinal Center B Cells

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Inguinal dLNs and spleens were prepared as described42 (link). Cells were transferred to 96 well V-bottom plates (Thermo Scientific, UK) and centrifuged (300 × g, 5 min). Supernatants were then discarded and cells were resuspended with addition of Fc Receptor Block (1:50) (eBioscience, UK) for 15 min. Following a wash in FACS buffer, cells were stained with anti-GL7 AlexaFluor 647 (eBioscience, UK) at 1:100 dilution, anti-CD95 PE (eBioscience, UK) at 1:100 dilution and anti-B220 PeCy7 (eBioscience, UK) at 1:200 for 30 min at RT in the dark. After this incubation, cells were washed twice and resuspended in FACS buffer and data were acquired on a LSRII (BD Bioscience, UK) and analyzed by FlowJo (TreeStar Inc, USA).

Li Y., Leneghan D.B., Miura K., Nikolaeva D., Brian I.J., Dicks M.D., Fyfe A.J., Zakutansky S.E., de Cassan S., Long C.A., Draper S.J., Hill A.V., Hill F, & Biswas S. (2016). Enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing Pfs25 to IMX313 multimerization technology. Scientific Reports, 6, 18848.

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Splenic Cell Immunophenotyping by Flow

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Splenic cells were incubated with the 2.4G2 anti-mouse FcγIII/II receptor monoclonal antibody (mAb) for 20 min at 4^oC, followed by incubation with a mixture of specific mAbs for 20 min at 4^oC. The following mAbs were used: anti-B220 PE-Cy7 (eBioscience, San Diego, CA, United States), anti-CD5 APC (eBioscience), and anti-CD11b PerCp-Cy5.5 (BioLegend, San Diego, CA, United States) mAb. Stained cells were analyzed using a FACSCanto flow cytometer (BD Biosciences).

Furuya Y., Kirimanjeswara G.S., Roberts S., Racine R., Wilson-Welder J., Sanfilippo A.M., Salmon S.L, & Metzger D.W. (2017). Defective anti-polysaccharide IgG vaccine responses in IgA deficient mice. Vaccine, 35(37), 4997-5005.

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Isolation and Characterization of Murine Lymphocytes

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Splenocytes were processed and stained according to standard protocols. Salivary glands were cut into small pieces and incubated in digestion buffer (PBS, Liberase TL at 0.04 WU/mL, 0.1% DNase) at 37 degrees for one hour in a shaker. The reaction was stopped by addition of RPMI 1640 supplemented with 10% FCS. Cells were then filtered and washed with PBS before further downstream applications. The following antibodies were used: anti-B220-PECy7, anti-GL7-FITC, anti-CD19-V450, CD45-FITC, CD8 PercpCy5.5 (eBiosciences) and CD4 APC. All antibodies were obtained from BD Biosciences except where indicated. Cells were analyzed on an LSR II flow cytometer (BD Biosciences) using FACSDiva software. Data were analyzed using Flowjo software.

Mahmoud T.I., Wang J., Karnell J.L., Wang Q., Wang S., Naiman B., Gross P., Brohawn P.Z., Morehouse C., Aoyama J., Wasserfall C., Carter L., Atkinson M.A., Serreze D.V., Braley-Mullen H., Mustelin T., Kolbeck R., Herbst R, & Ettinger R. (2016). Autoimmune manifestations in aged mice arise from early-life immune dysregulation. Science translational medicine, 8(361), 361ra137.

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Characterization of Arthritic Knee Synovial Cells

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Both knee joints from arthritic mice were dissected carefully without disintegrating the femur or tibia. Muscle tissue was removed from the femur and tibia. Afterwards, knee joints were cracked open and digested using a collagenase (1.2 mg/mL, C8051)/dispase II (25 U/mL, D4693) (both from Sigma-Aldrich) solution for 30 minutes at 37°C. The digestion was stopped by adding 10% FCS and the solution was passed through a 50-μm mesh. Synovial cells were washed twice with PBS/2% FCS, blocked with anti-rat IgG and CD16/CD32, and subsequently stained with anti-CD11b-FITC (1:800), anti-CD45-PE (1:200), anti-B220-PE-Cy7 (1:500), anti-CD3-APC (1:200), and an anti-Gr1-A700 (1:100) antibody for 30 minutes at 4°C in the dark (all antibodies were purchased from eBioscience, Mannheim, Germany). Thereafter, cells were washed twice with PBS and analyzed with the BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA) and the FlowJo vX software (Tree Star Inc., Ashland, OR, USA).

Albus E., Sinningen K., Winzer M., Thiele S., Baschant U., Hannemann A., Fantana J., Tausche A.K., Wallaschofski H., Nauck M., Völzke H., Grossklaus S., Chavakis T., Udey M.C., Hofbauer L.C, & Rauner M. (2015). Milk Fat Globule-Epidermal Growth Factor 8 (MFG-E8) Is a Novel Anti-inflammatory Factor in Rheumatoid Arthritis in Mice and Humans. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 31(3), 596-605.

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