Ra3 6b2

Manufactured by BD

Sourced in United Kingdom, Switzerland

The RA3-6B2 is a laboratory equipment designed for specific applications. It has a core function of performing tasks relevant to its intended use. However, without additional details about the product, I cannot provide a more detailed description while maintaining an unbiased and factual approach. Description not available.

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Lab products found in correlation

30 protocols using ra3 6b2

CD8/CD4 T Cell Purification and Activation

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CD8 T cells were purified from the spleens of adult P14 mice via negative selection by antibody depletion of B220 (RA3-6B2, BD Pharmingen), CD11c (HL3, BD Pharmingen), CD11b (M1/70, BD Pharmingen), CD16/32 (2.4G2, BD Pharmingen), IA/IE (2G9, BD Pharmingen) and CD4 (L3T4 RM4-5, BD Pharmingen) with magnetic bead separation (anti-rat IgG Dynal beads, Invitrogen). CD4 T cells were purified from the spleens of adult Smarta mice via negative selection by antibody depletion of B220 (RA3-6B2, BD Pharmingen), CD11b (M1/70, BD Pharmingen), CD16/32 (2.4G2, BD Pharmingen), IA/IE (2G9, BD Pharmingen), CD8α (Clone 53–6.7, BD Pharmingen) with magnetic bead separation (anti-rat IgG Dynal beads, Invitrogen). Cells were counted and labeled with CellTrace^TM Violet proliferation dye (Invitrogen) according to manufacturers instructions. FACS sorted DCs were plated at 1×10⁴ cells/well of a round bottom 96 well plate together with 1×10⁵ cells/well of purified CD8 T cells or 3×10⁴ CD4 T cells in RPMI 1640 (Invitrogen) supplemented with 5 mM Hepes, 10% FBS (HyClone), 1% Penicillin Streptomycin (Life Technologies), 1% Glutamax (Life Technologies) and 50μM ≤β-mercaptoethanol (Life Technologies). As positive control for T cell proliferation, T cells were co-cultured with GP33 or GP61 (Abgent) peptide pulsed naïve CD8α^neg DCs.

Baca Jones C., Filippi C., Sachithanantham S., Rodriguez-Calvo T., Ehrhardt K, & von Herrath M. (2014). Direct Infection of Dendritic Cells during Chronic Viral Infection Suppresses Antiviral T Cell Proliferation and Induces IL-10 Expression in CD4 T Cells. PLoS ONE, 9(3), e90855.

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Tissue Immunophenotyping by Antibody Staining

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Tissue isolation and slide preparation were performed as previously described [18 (link)]. Tissue sections were stained with antibodies specific for CD4 (L3T4; R&D Systems), CD169 (c3D6.112; AbDSerotec), MARCO (ED31; AbDSerotec), B220 (RA3-6B2; BD), and F4/80 (BM8; Ebioscience).

Hodge D.L., Berthet C., Coppola V., Kastenmüller W., Buschman M.D., Schaughency P.M., Shirota H., Scarzello A.J., Subleski J.J., Anver M.R., Ortaldo J.R., Lin F., Reynolds D.A., Sanford M.E., Kaldis P., Tessarollo L., Klinman D.M, & Young H.A. (2014). IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice. Journal of autoimmunity, 53, 33-45.

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Characterizing Qβ-specific Plasma Cells Post Adoptive Transfer

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For FCM staining spleens of mice after adoptive transfer were isolated in RPMI supplemented with 2% FCS and antibiotics and a single cell suspension was prepared. Red blood cells were lysed using ACK buffer prior to staining. Fc receptors were blocked using an anti-CD16/32 antibody (2.4G2, BD). To discriminate Qβ-specific plasma cells (PCs) from Qβ-specific activated and CS B cells, surface immunoglobulins (Ig) of specific cells were blocked using unlabelled Qβ VLPs. PCs were further stained with and characterized as IgM (polyclonal, Jackson ImmunoResearch), IgD (11-26c (11-26), eBioscience), CD4 (H129.19, BD), CD8 (53-6.7, BD), GR1 (RB6-8C5, BD), CD11b (M1/70, BD), CD11c (HL3, BD) negative (all antibodies labeled with PE), and B220-PE-Cy7 (RA3-6B2, BD) low. To detect Qβ specific PCs by intracellular staining of specific Ig, splenocytes were permeabilized using FACS lysing solution (BD, 349202) containing 0.04% Tween20 and stained with Alexa Flour 488 labeled Qβ VLPs. The congenic marker Ly5.1 (antibody labeled with APC, A20, eBioscience) identified all transfer derived B cells.
Qβ VLPs were labeled with the Alexa Flour 488 protein labeling kit (Thermo Fisher Scientific, A10235) according to the manufacturer's instructions.

Krueger C.C., Thoms F., Keller E., Leoratti F.M., Vogel M, & Bachmann M.F. (2019). RNA and Toll-Like Receptor 7 License the Generation of Superior Secondary Plasma Cells at Multiple Levels in a B Cell Intrinsic Fashion. Frontiers in Immunology, 10, 736.

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Histological and Immunohistochemical Analysis of Mouse Organs

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Histological analysis of mouse organs was performed on 4 μm thick FFPE tissue sections, stained with Hematoxylin & Eosin (H&E) (Thermo Scientific)^{53 (link)}. The following primary antibodies and dilutions were used for immunohistochemical analysis: rabbit polyclonal anti-Bcl6 (1:300) (N3, Santa Cruz Biotechnology) and anti-Pax5 (1:400) (Neomarker); rabbit monoclonal anti-CD3 (1:800) (clone SP7, Neomarker) and anti-Ki67 (1:200) (clone SP6, Thermo Scientific); rat monoclonal anti-B220 (1:400) (clone RA3-6B2, BD Biosciences) and anti-CD138 (1:200) (clone 281-2, BD Biosciences); mouse monoclonal anti-BCL2 (1:200) (clone Bcl-2/100, BD Biosciences).

Zhang J., Dominguez-Sola D., Hussein S., Lee J.E., Holmes A.B., Bansal M., Vlasevska S., Mo T., Tang H., Basso K., Ge K., Dalla-Favera R, & Pasqualucci L. (2015). Disruption of KMT2D perturbs germinal center B cell development and promotes lymphomagenesis. Nature medicine, 21(10), 1190-1198.

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Multiparameter Flow Cytometry Assay

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Cells were resuspended in PBS containing 1% FCS and 0.05% sodium azide and incubated with an optimal dilution of fluorochrome conjugated antibodies. Antibodies employed included those specific for B220 (RA3-6B2, BD), IgM (b-7-6), IgD (11-26c, Southern Biotech), IgM^a (RS3.1), SHIP-1 (polyclonal rabbit antibody against the C-terminal 100aa), CD138 (281-2, BD), CD23(EBVCS-5, Biolegend), CD21 and D1.3 anti-HEL recognizing a distinct, non-hindering HEL epitope [20 (link)]. The D1.3 hybridoma was obtained from Richard Willson (Univ. of Houston ) via National Cell Culture Center (www.nccc.com/). Antibodies to SHIP-1, CD21, IgM^a, IgM and HEL (D1.3) were produced in our own laboratory and were directly conjugated to Alexa (Invitrogen) or DyLight (Pierce) fluorochromes, according to the manufacturer’s protocol. For detection of plasmablasts, splenocytes were first stained with B220-FITC and CD138PE, then fixed and permeabilized with BD Cytofix/Cytoperm^™ and stained with HEL-DyLight 650 (HEL from Sigma reconstituted and conjugated to DyLight-650). Cells that were highly stained with HEL-DyLight 650 were routinely found to be CD138 positive (data not shown). Cells were analyzed on a Cyan (Beckman Coulter) or LSRII flow cytometer (BD) with data analysis using FlowJo (Tree Star).

Akerlund J., Getahun A, & Cambier J.C. (2015). B cell expression of the SH2-containing inositol 5-phosphatase (SHIP-1) is required to establish anergy to high affinity, proteinacious autoantigens. Journal of autoimmunity, 62, 45-54.

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Immunohistochemical Analysis of Lungs

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Lungs were fixed in formalin for histologic analysis or used for bronchial alveolar lavage (BAL) collection (18 (link)). Sections were processed for H&E staining or immunohistochemistry staining with antibodies to CPT1 (8F6AE9 Abcam; 1:100) or HADHA (ab54477 Abcam; 1:500). Some sections were also stained with antibodies to CPT1 or HADHA together with F4/80 (SP115 Abcam; 1:100), Ly-6G (RB6–8C5 ebioscience; 1:500), Siglec-F (E50–2440 BD Biosciences; 1:500), or CD45R (RA3–6B2; BD Biosciences; 1:500). Protocols were described previously (7 (link)). BAL fluids were subjected to cytospin and stained with Diff-Quik (IMEB).

Al-Khami A.A., Ghonim M.A., Valle L.D., Ibba S.V., Zheng L., Pyakurel K., Okpechi S.C., Garay J., Wyczechowska D., Sanchez-Pino M.D., Rodriguez P.C., Boulares A.H, & Ochoa A.C. (2017). Fueling the Mechanisms of Asthma: Increased Fatty Acid Oxidation in Inflammatory Immune Cells May Represent a Novel Therapeutic Target. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 47(9), 1170-1184.

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Lung Cell Isolation and Characterization

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Harvested organs were
cut into small pieces and incubated for 1 h at 37 °C with a mix
of DNase I (100 μg/mL, Sigma-Aldrich) and collagenase D (400
U/mL, Roche). Lung cells were washed and filtered before being incubated
with saturating doses of purified 2.4G2 (anti-mouse Fc receptor, ATCC)
in 200 μL of PBS, 0.2% BSA, and 0.02% NaN₃ (FACS
buffer) for 20 min at 4 °C to prevent antibody binding to Fc
receptors. Various fluorescent monoclonal antibody (mAb) combinations
in FACS buffer were used to stain (3–5) × 10⁶ cells. Acquisitions were done on a FACScanto II cytofluorometer
(Becton Dickinson) with the following mAbs: fluorescein (FITC)-coupled
anti-CD3 (145-2C11, BD Biosciences), FITC-coupled anti-CD11c (HL3,
ThermoFisher), FITC-coupled anti LY6G (1A8, BD Biosciences), phycoerythrine
(PE)-coupled anti-SiglecF (E50-2440, BD Biosciences), PE-coupled anti-MHCII
(M5, BD Biosciences), PE-coupled anti CD11b (M1/70, BD Biosciences),
allophycocyanin (APC)-coupled anti-F4/80 (BM8, BD Biosciences), APC-coupled
anti-B220 (RA3-6B2, BD Biosciences), APC-coupled anti-CD11c (HL3,
BD Biosciences), Brillant violet 421 (BV421)-coupled anti SiglecF
(E50-2440, BD Biosciences), BV421-coupled anti-MHCII (M5, BD Biosciences).
Fixable viability dye aqua (ThermoFisher) was used to gate viable
cells.

Machelart A., Salzano G., Li X., Demars A., Debrie A.S., Menendez-Miranda M., Pancani E., Jouny S., Hoffmann E., Deboosere N., Belhaouane I., Rouanet C., Simar S., Talahari S., Giannini V., Villemagne B., Flipo M., Brosch R., Nesslany F., Deprez B., Muraille E., Locht C., Baulard A.R., Willand N., Majlessi L., Gref R, & Brodin P. (2019). Intrinsic Antibacterial Activity of Nanoparticles Made of β-Cyclodextrins Potentiates Their Effect as Drug Nanocarriers against Tuberculosis. ACS Nano, 13(4), 3992-4007.

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Immunofluorescence Staining of OSCC

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Frozen sections were fixed in 2% paraformaldehyde/PBS pH 7.4 and blocked with 10% goat serum, 2% BSA, 0.02% fish skin gelatin and 0.05% TritonX100 (Sigma) in PBS for 1 h at room temperature. Paraffin sections of human OSCC were subjected to heat-mediated antigen retrieval (citrate buffer, pH6) prior to blocking. Primary antibodies were incubated overnight at 4 °C, followed by 1 h incubation at room temperature in secondary antibody.
The following primary antibodies were used: Foxp3 (eBioscience, clone FJK-16s, 1/100, and Abcam, clone 236 A/E7, 1/50), anti-Loricrin, anti-Krt76 (Santa Cruz, clone F-12, 1/100, and Sigma, HPA019656, 1/100), Krt14 (Covance, PRB-155P, 1/1000), B220/CD45R (eBioscience, clone RA3-6B2, 1/100), CD3 (BD Pharmingen, clone 17A2, 1/150), CD45 (BD Pharmingen clone 30-F11, 1/150); and secondary antibodies: anti-goat, anti-mouse and anti-rabbit Alexa Fluor 488, 568 and 633 (Life Technologies, 1/300).
EdU staining was performed with a Click-it EdU imaging kit (Life Technologies) according to the manufacturer’s recommendations. DAPI (Life Technologies) was used as a nuclear counterstain. Slides were mounted using ProLong Gold anti-fade reagent (Life Technologies). Images were acquired with a Nikon A1 Upright Confocal microscope. Images were analysed using ICY image analysis software^{55 (link)}.

Sequeira I., Neves J.F., Carrero D., Peng Q., Palasz N., Liakath-Ali K., Lord G.M., Morgan P.R., Lombardi G, & Watt F.M. (2018). Immunomodulatory role of Keratin 76 in oral and gastric cancer. Nature Communications, 9, 3437.

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Multiparameter Flow Cytometry of Immune Cells

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PE-conjugated anti-CD23 (eBioscience-B3B4), FITC-conjugated anti-IgM (Sigma-μ chain specific) or brilliant violet (BV) 421-conjugated anti-IgM (BD pharmingen-R6-60.2), APC-conjugated anti-CD19 (BD bioscience-1D3), FITC-conjugated anti-CD3ε (Immunotools-145-2C11), APC-conjugated anti-CD11b (Biolegend- M1/70) and PE-conjugated anti-Gr-1(Immunotools- RB6-8C5) were used for flow cytometric analysis of blood leukocytes. Biotinylated anti-B220 (eBioscience-RA3-6B2) or Alexa 647-conjugated anti-B220 (BD Pharmingen- RA3-6B2), PE-conjugated anti-IgD (Biolegend-clone 11-26c.2a), FITC-conjugated anti-IgM (Sigma-μ chain specific) or BV 421-conjugated anti-IgM (BD Pharmingen- R6-60.2), FITC- conjugated anti-CD21 (BD Pharmingen- eBio8D9), PE-conjugated anti-CD23 (eBioscience-B3B4), APC-conjugated anti-CD19 (BD bioscience-1D3) or Alexa 700-conjugated anti-CD19 (BD Pharmingen-1D3) were used for flow cytometric analysis of BM or spleen cells. Biotinylated antibodies were detected with APC-conjugated streptavidin (Immunotools) or BV 605-conjugated streptavidin (BioLegend). Rat anti-mouse SIRPα (mAb P84; rat IgG1; a generous gift from Dr.Carl Lagenaur, university of Pittsburgh) was purified and conjugated to Alexa 488 as previously described [18 (link)].

Kolan S.S., Lejon K., Koskinen Holm C., Sulniute R., Lundberg P., Matozaki T, & Oldenborg P.A. (2015). Non-Hematopoietic and Hematopoietic SIRPα Signaling Differently Regulates Murine B Cell Maturation in Bone Marrow and Spleen. PLoS ONE, 10(7), e0134113.

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Histological and Immunohistochemical Analysis of Mouse Organs

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