TCL1-192 cells were enumerated by incubation with fluorescein isothiocyanate anti-CD45R/B220 (553087), Phycoerythrin anti-CD5 (553023), APC-CXCR4 (558644), PerCPCy5.5-CXCR5 (560528), Phycoerythrin-CD49d (553157; BD Biosciences, San Jose, CA, USA), PECy7 anti-CD39 (25-0391), eFluor 450 anti-CD69 (48-0691), A700 anti-CD62L (56-0621), PECy7 anti-CD5 (25-0051; eBioscience, San Diego, CA, USA), APC anti-S1PR1 (R&D, Minneapolis, MN, USA, FAB7089A) and PECy7 anti-CD29 (BioLegend, San Diego, CA, USA, 102222). BrdU incorporation assays were done following the manufacture's protocol (BD Biosciences). Flow cytometry data were obtained with a BD LSRII machine (BD Biosciences), and analyzed with FlowJo software (FlowJo, LLC, Ashland, OR, USA).
Anti cd45r b220
Manufactured by BD
Sourced in United States
Anti-CD45R/B220 is a monoclonal antibody that recognizes the CD45R (B220) isoform of the CD45 antigen, which is expressed on the surface of B cells and a subset of T cells. It is commonly used in flow cytometry and immunohistochemistry applications for the identification and enumeration of B cells.
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Lab products found in correlation
Anti cd3, by Santa Cruz Biotechnology (3 mentions)
Anti cd11b, by BD (3 mentions)
Anti cd4, by BD (3 mentions)
Fc block, by BD (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Axioplan 2 microscope, by Zeiss (2 mentions)
Clone ra3 6b2, by BD (2 mentions)
Anti cxcl13, by R&D Systems (2 mentions)
Endogenous biotin, by Merck Group (2 mentions)
Clone m 20, by Santa Cruz Biotechnology (2 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Anti ter119, by BD (2 mentions)
Cxcr4 apc, by BD (1 mentions)
Lsrii machine, by BD (1 mentions)
Percpcy5.5 cxcr5, by BD (1 mentions)
Efluor 450 anti cd69, by Thermo Fisher Scientific (1 mentions)
A700 anti cd62l, by Thermo Fisher Scientific (1 mentions)
Pecy7 anti cd29, by BioLegend (1 mentions)
Biotinylated secondary antibody, by BD (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
11 protocols using anti cd45r b220
1
Immunophenotyping of TCL1-192 Cells
2
Lung Immunofluorescent Staining Protocol
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Lung lobes were perfused with 10% neutral-buffered formalin and embedded in paraffin. For immunofluorescent staining, formalin-fixed lung sections were cut, immersed in xylene to remove paraffin, and then sequentially hydrated in absolute ethanol, 95% ethanol, 70% ethanol and water. Antigens were unmasked with a DakoCytomation Target Retrieval Solution (Dako, Carpinteria, CA) and non-specific binding was blocked with 5% (v/v) normal donkey serum and Fc block (BD Pharmingen). Endogenous biotin (Sigma-Aldrich) was neutralized by adding avidin followed by incubation with biotin. Sections were probed with anti-CD45R/B220 to detect B cells (Clone RA3-6B2, BD Pharmingen), anti-CXCL13 (AF470, R&D systems) and anti-CD3 to detect T cells (Clone M-20, Santa Cruz Biotechnology, Santa Cruz, CA). B-cell follicles were outlined with the automated tool of the Zeiss Axioplan 2 microscope (Carl Zeiss, Thornwood, NY) and average size in squared microns was calculated as described before12 (link).
3
Immunohistochemical Staining of Mouse Brain
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Mice were anesthetized with a lethal dose of sodium pentobarbital and trans-cardially perfused with PBS. For frozen sections, mice were subsequently perfused and brains were isolated in 4% paraformaldehyde (PFA) followed by 20% sucrose and then flash frozen in liquid nitrogen. 10 μm horizontal or sagittal sections were cut and mounted on Superfrost Plus (Fisher Scientific) glass slides. For paraffin sections, fresh brains were transferred into 10% phosphate-buffered formalin followed by 70% EtOH and paraffin embedding. 5 μm sections were mounted onto glass slides. Sections were de-paraffinized and antigen retrieval was performed in sodium citrate buffer. Sections were incubated with 3% hydrogen peroxide and blocked in 5% serum for 1 hr at room temperature (RT). Slides were incubated with primary antibody (anti-CD3ε [BD Biosciences], anti-CD45R/B220 [BD Biosciences], or anti-VSV(G) [a gift from Dr. E. Brown, University of Ottawa]) for 2 hr, followed by a biotinylated secondary antibody (BD Biosciences) for 30 min at RT. Avidin-Biotin-Peroxidase (ABC Kit, Vector Laboratories) was applied as per the manufacturer’s protocol. 3,3'-Diaminobenzidine (DAB; Sigma) was added for 2 min before immersing in ddH2O and counterstaining with hematoxylin, followed by slide mounting in xylene medium. Sections were scanned using a UMAX Astra 1220S scanner.
4
CD4+ and CD8+ T Cell Isolation and Stimulation
5
Enrichment of Splenocyte Subsets
6
Pten-Deficient Mouse Model of Leukemia
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Animals were housed in the St Jude Animal Resources Center with access to sterilized food and water ad libitum, and all experiments were approved by the Institutional Animal Care and Use Committee of St. Jude Children’s Research Hospital. B6.129S4fltm1Hwu/J (Ptenfl/fl) mice were kindly provided by Dr. Suzanne Baker. ETV7Tg+/WT on the 129/CL57/B6 (129SvEv;C57Bl/6 mixed genetic background) were generated by backcrossing ETV7Tg+/WT FVB mice onto wild-type 129/CL57/B6 mice for more than 10 generations prior to crossing them with Ptenfl/fl;Mx1-Cre. 4–6-Week-old Ptenfl/fl;Mx1-Cre and Ptenfl/fl;Mx1-Cre;ETV7Tg+/WT mice were injected intraperitoneally with seven doses of polyinosine–polycytidine (pIpC) (25 μg/g) every other day for 14 days to induce Cre expression as described previously (Yilmaz et al. 2006 (link)). Five days after pIpC injection, total BM was harvested and analyzed by colony-forming cell assays. The pIpC treated mice were observed daily and moribund mice were euthanized by CO2 inhalation. For pathologic diagnosis, the spleen, thymus, and sternum of sick mice were fixed in 10% neutral-buffered formalin and embedded in paraffin. All sections were stained with hematoxylin and eosin (H&E), and immunostained with anti-CD3 (Santa Cruz), anti-CD45R/B220 (BD Biosciences), and anti-myeloperoxidase (MPO; Dako) antibodies.
7
Immunohistochemical Evaluation of Vaccine Response
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For immunohistochemical evaluation, mice were euthanized at 3, 6, 24, 48, 72, and 96 hours after vaccination and the skin and axillary, inguinal, and popliteal lymph nodes were collected, fixed in PFA 1%, and embedded in OCT and tissue sections were stained with hematoxylin and eosin (H&E) for histological examination. Skin and lymph nodes sections were incubated with the following primary antibodies: rat mAb anti-CD4, anti-CD8α, anti-CD11b, anti-CD45R/B220, anti-Gr-1 (BD Pharmingen, Milan, Italy), anti-CD68 (Abcam, Cambridge, UK), and anti-CD11c (Chemicon International, USA). After washing, slides were overlaid with appropriate secondary antibodies. Immunostaining was developed with Vulcan Fast Red (Biocare, Milan, Italy) alkaline phosphatase method. For immunofluorescence, secondary antibodies conjugated with Alexa 488 and Alexa 546 (Invitrogen, Life Technologies, Monza, Italy) were used. Nuclei were stained with DRAQ5 (Alexis, Life Technologies, Monza, Italy), YO PRO-3, or TO PRO-3 (Molecular Probes, Monza, Italy). Image acquisition was performed using Zeiss LSM 510 Meta confocal microscope (Carl Zeiss SpA, Milan Italy).
The slides were examined in a double-blind fashion, and digital images of representative areas were taken.
The slides were examined in a double-blind fashion, and digital images of representative areas were taken.
8
Isolation of Alveolar Macrophages from BAL
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BAL was performed as previously described22 (link). The immunomagnetic separation system (BD Biosciences Pharmingen, San Diego, CA) was used to isolate AM from BAL fluid. Magnetic nanoparticle-conjugated antibodies (anti-mouse Gr-1, anti-CD4, anti-CD8, and anti-CD45R/B220 antibodies; BD Biosciences Pharmingen, San Diego, CA) were chosen to label and remove PMN and lymphocytes. The resulting cells consisted of >98% macrophages, and cell viability was >95%.
9
Multiparametric Flow Cytometry Analysis of Immune Cell Subsets
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Single-cell suspensions of blood, BM, lymph nodes, spleen and thymus were prepared. Lymph nodes, splenocytes and thymocytes were passed through a 40-μm cell strainer. Erythrocytes were lysed with ammonium chloride. Cells were washed twice and stained for 20 min at 4°C in PBS/0.5 mM EDTA/0.5% BSA with the following rat mAbs from BD Biosciences: anti-H2kd (SF1-1.1-FITC and PE), anti-CD8a (53-6.7-APC), anti-Ly-6G and Ly6C/Gr1 (RB6-8C5-APC), anti-CD45R/B220 (RA3-6B2-PerCP-Cy5.5), anti-CD25 (PC61-PerCP-Cy5.5), anti-CD4 (RM4-5-PE-Cy7), anti-CD80 (16-10A1-PE), anti-CD86 (GL1-PerCP-Cy5.5), anti-CD11c (HL3-PE-Cy7), anti-CD3e (145-2C11-APC-Cy7) and anti-CD11b (M1/70-APC-Cy7). For staining of regulatory T-cells, Anti-Mouse/Rat FoxP3 Staining Set APC (eBioscience, San Diego, CA, United States) was used following the manufacturer’s instructions. Samples were analyzed by BD FACS Canto II (BD Biosciences) and FlowJo 7.6.5 Software (TreeStar Inc., Ashland, OR, United States).
10
Histological Analysis of Axillary Lymph Nodes and Skin
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Serial sections (5 μm thick) of the axillary lymph node were obtained and stained with hematoxylin and eosin. In addition, in the axillary lymph node, macrophages, B and T lymphocytes were detected using an anti-Iba-1 (019-19741, WAKO, United States; RRID AB_839504), an anti-CD45R/B220 (BD Pharmingen, United States; RRID AB_396673) and an anti-CD3 epsilon (Sc-1127, Santa Cruz Biotechnology, United States; RRID AB_631128), respectively. Cryosections (7 μm thick) from OCT embedded organs were stained with ORO to detect neutral lipid accumulation. Lipid deposition was measured as the percent of ORO positive area over the total area.30 (link)The Aperio ScanScope GL Slide Scanner (Aperio Technologies, CA) was used to acquire digital images that were subsequently processed with the ImageScope software. Two operators blinded to mouse genotypes quantified all histological parameters.
Skin semithin sections, 2 µm thick, were stained with toluidine blue and observed with a Nikon Eclipse E600 microscope equipped with a Nikon digital camera DXM1200 (Nikon, Tokyo, Japan). Ultrathin sections were obtained with an Ultracut ultramicrotome (Reichert Ultracut R-Ultramicrotome, Leika, Wien, Austria) and stained with uranyl acetate/lead citrate before observation by a Jeol CX100 transmission electron microscope (Jeol, Tokyo, Japan).
Skin semithin sections, 2 µm thick, were stained with toluidine blue and observed with a Nikon Eclipse E600 microscope equipped with a Nikon digital camera DXM1200 (Nikon, Tokyo, Japan). Ultrathin sections were obtained with an Ultracut ultramicrotome (Reichert Ultracut R-Ultramicrotome, Leika, Wien, Austria) and stained with uranyl acetate/lead citrate before observation by a Jeol CX100 transmission electron microscope (Jeol, Tokyo, Japan).
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