Cd19 ebio1d3

Manufactured by Thermo Fisher Scientific

CD19 (eBio1D3) is a mouse monoclonal antibody that recognizes the CD19 antigen. CD19 is a member of the immunoglobulin superfamily and is expressed on the surface of B cells, B cell precursors, and some non-Hodgkin's lymphomas. The CD19 antibody can be used for the identification and analysis of B cells and B cell-related disorders in flow cytometry applications.

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Close spelling variants of this product

EBio1D3 (13 citations) CD19 (clone eBio1D3), (3 citations) Anti-CD19–PE-Cy7 (clone eBio1D3 (1D3), (2 citations) CD19 PE (eBio1D3) (2 citations)

7 protocols using cd19 ebio1d3

Spleen and Heart Cell Isolation and Characterization

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Single cell suspensions from spleens and lymph nodes were obtained via passing through 70 μm cell strainers in cold PBS^−/−. Hearts were collected and digested with Liberase TM (Roche). Erythrocytes were removed with lysis buffer (BD Biosciences) from spleen and heart cell suspensions. Cells were stained at the following dilutions of stock reagents: Live/dead Aqua Fluorescent Reactive Dye 1:1,000 (Life Techonologies), anti-mouse CD16/32 1:100 (2.4G2, BD Pharmigen), anti-mouse 1:100 CD45 (30-F11, eBioscience), anti-mouse 1:100 CD3ɛ (145-2C11, BioLegend), anti-mouse 1:100 CD19 (eBio1D3, eBioscience), anti-mouse 1:100 CD11b (M1/70, Biolegend), 1:100 CD11c (Bu15, eBioscience), F4/80 1:100 (CI:A3-1, Serotec) in Supplementary Fig. 5d, F4/80 1:100 (BM8, eBioscience) in Fig. 5d,e and Supplementary Fig. 5c, IL-10 1:80 (JES5-16E3, eBioscience), FoxP3 1:100 (FJK-165, eBioscience), Ly6C 1:100 (HK1.4, eBioscience) or anti-CD25 1:100 (PC61.5, eBioscience). An eBioscience intracellular staining kit was used were applicable. Samples were acquired on a fluorescence-activated cell sorting Canto II (BD) and analysed with FlowJo10.

Kallikourdis M., Martini E., Carullo P., Sardi C., Roselli G., Greco C.M., Vignali D., Riva F., Ormbostad Berre A.M., Stølen T.O., Fumero A., Faggian G., Di Pasquale E., Elia L., Rumio C., Catalucci D., Papait R, & Condorelli G. (2017). T cell costimulation blockade blunts pressure overload-induced heart failure. Nature Communications, 8, 14680.

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Multiparametric Flow Cytometry of Immune Cells

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Single-cell suspensions of bone marrow, spleen, and lymph nodes were incubated with 1 x ACK solution (for red blood cell lysis) to deplete erythrocytes. Staining was performed with the following antibodies (conjugated with FITC, PE, APC, eFI450, BV786, AF700, PE-Cy7, PerCP Cy5.5, APC-eFI780 or biotin): anti–Siglec-H (551.3D3; Biolegend), anti-mPDCA1 (JF05-1C24.1; Biolegend), anti-CD11c (N418; Biolegend), anti-B220 (RA3-6B2; Biolegend), anti-CCR9 (242503; R&D Systems), anti-FcgR2b (own hybridoma), anti-IL2r (PC61; Biolegend), CD19 (eBio1D3, eBioscience), CD3 (17A2, eBioscience), CD5 (53-7.3, eBioscience), CD4 (GK1.5, eBioscience), CD138 (281-2, BD), CD95 (Jo2, BD), IgD (11-26c.2a, BD), CD80 (16-10A1, eBioscience), PD-L2 (TY25, BD), IL-4 (11B11, BD), IL-6 (MP5-20F3, BD), IL-10 (JES5-16E3, eBioscience), IFN-g (XMG1.2, eBioscience) and Fc-block (2.4G2; own hybridoma). Biotinylated antibodies were detected using streptavidin PerCPCy5.5 or streptavidin PECy7 (Biolegend). Cells were analysed using either Cytoflex (Beckmann Coultier) and FlowJo software (Tree Star).

Szumilas N., Corneth O.B., Lehmann C.H., Schmitt H., Cunz S., Cullen J.G., Chu T., Marosan A., Mócsai A., Benes V., Zehn D., Dudziak D., Hendriks R.W, & Nitschke L. (2021). Siglec-H-Deficient Mice Show Enhanced Type I IFN Responses, but Do Not Develop Autoimmunity After Influenza or LCMV Infections. Frontiers in Immunology, 12, 698420.

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Placental Immune Cell Isolation

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Pregnant dams were euthanized before hysterectomy. Individual placentas were dissected from the uterus with the removal of the decidua before manual dissociation and digestion in a solution of Hanks’ Balanced Salt Solution + 5% fetal bovine serum (FBS), 1% 0.5-M CaCl₂, and 10 mg/mL of collagenase P. The placentas were digested at 37°C for 15 minutes and quenched with Hanks’ Balanced Salt Solution + 10% FBS. Splenocytes were collected by manual dissociation of the spleen. Placental cells or splenocytes were stained with the following antibodies: CD45 (30-F11), B220 (RA3–6B2), CD8 (53–6.7), H2K^d (SF1–1.1), CD21 (7G6), CD49b (DX5), and CD31 (MEC13.1) purchased from BD Bioscience (Franklin Lakes, NJ); H2K^b (AF6–88.5.5.3), Foxp3 (FJK-16S), immunoglobulin M (II/41), CD23 (B3B4), CD3 (17A2), TCRβ (H57–597), CD19 (eBio1D3), and CD11B (M1.70) purchased from eBioscience (San Diego, CA); and CD4 (RM4–4), CD19 (6D5), and CD268 (7H22-E16) purchased from BioLegend (San Diego, California). The eBioscience Foxp3 Transcription Factor Staining Kit (#00–5523-00) was used for the detection of Foxp3. Splenocytes were used as compensation controls.

McNew K.L., Abraham A., Sack D.E., Smart C.D., Pettway Y.D., Falk A.C., Lister R.L., Faucon A.B., Bejan C.A., Capra J.A., Aronoff D.M., Boyd K.L, & Moore D.J. (2022). Vascular alterations impede fragile tolerance to pregnancy in type 1 diabetes. F&S science, 3(2), 148-158.

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Multiparameter Flow Cytometric Analysis of Mouse B Cells

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Monoclonal antibodies to the following mouse E-proteins were used in multiparameter flow cytometric analysis: B220/CD45R (RA3-6B2), CD19 (eBio1D3), CD86 (GL1), CXCR4/CD184 (2B11), and GL7 (GL7) from eBioscience; CD138/Syndecan-1 (281-2), Fas/CD95 (Jo2), and IgG1 (X56) from BD; and peanut agglutinin (PNA) from Vector Laboratories. Antibodies to the following proteins were generated and conjugated in-house: Bcl6 (7D1-10), CD21 (7G6), CD23 (B3B4), CD38 (NIMR5), CD138/Syndecan-1 (7/11-8B8), Gr1/Ly6c (RB6-8C5), IgD (11-26C), and IgM (331.12). All antibodies were titrated for optimum concentration before use, with dilutions ranging from 1/100 to 1/1,600. FcγRII/III (24G2; supernatant) was used for blocking. Biotinylated monoclonal antibodies were detected with streptavidin conjugated to PECy7 (BD). NP was conjugated in-house. Viable cells were identified by propidium iodide or SytoxBlue (Invitrogen) exclusion. For intracellular staining, cells were fixed, permeabilized, and stained using the reagents and protocol in the Foxp3 Staining Buffer Set (eBioscience). Cells were analyzed on FACSCanto II or LSR Fortessa cytometers (BD). Cells were sorted on a FACS Aria cytometer (BD). Data were processed using FlowJo software.

Gloury R., Zotos D., Zuidscherwoude M., Masson F., Liao Y., Hasbold J., Corcoran L.M., Hodgkin P.D., Belz G.T., Shi W., Nutt S.L., Tarlinton D.M, & Kallies A. (2016). Dynamic changes in Id3 and E-protein activity orchestrate germinal center and plasma cell development. The Journal of Experimental Medicine, 213(6), 1095-1111.

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Multiparametric Flow Cytometry Analysis

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Cell suspensions were stained with antibodies for mouse studies as follows: FITC-conjugated B220 (RA3-6B2, eBioscience), CD3 (17A2, eBioscience), CD4 (RM4-5, eBioscience), CD5 (53–7.3, eBioscience), CD8α (53–6.7, eBioscience), CD11b (M1/70, eBioscience), CD11c (N418, eBioscience), CD19 (eBio1D3, eBioscience), Gr-1 (RB6-8C5, eBioscience), TCRβ (N57-597, eBioscience), Ter-119 (Ter119, eBioscience), γδTCR (eBioGL3, eBioscience), NK1.1 (PK136, eBioscience), FcεR1 (MAR-1, eBioscience). PE-conjugated ST2 (DJ8, MD Biosciences), PerCP/Cy5.5-cinjugated CXCR4 (Biolegend). APC-conjugated CD90.2 (53–2.1, eBioscience). Alexa Fluor 700-conjugated CD45 (30-F11, eBioscience). BV421-conjugated Ly-6A/E (D7, BD Biosciences). PE-Cyanine 7-conjugated KLRG1 (2F1, BD Biosciences).
To stain GRK2 antigen (R&D systems), cells were fixed and permeabilized after cell surface marker staining. Cells were analyzed by BD LSRII flow cytometer (BD Bioscences) and FlowJo software.

Lai D., Chen W., Zhang K., Scott M.J., Li Y., Billiar T.R., Wilson M.A, & Fan J. (2022). GRK2 regulates group 2 innate lymphoid cell mobilization in sepsis. Molecular Medicine, 28, 32.

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Multiparameter Flow Cytometry for Analyzing Lymphocyte Populations

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For analysis of lymphocyte populations, single-cell suspensions were prepared from homogenised spleens or bone marrow. For splenic suspension preparation, erythrocytes were destroyed with lysis buffer (BD Biosciences). The cells were treated with the appropriate combination of the following antibodies: CD16/32 (Fc block) (93) (eBioscience), B220 (RA3-6B2) (eBioscience), CD19 (eBio-1D3) (eBioscience), IgD (11-26c.2a) (eBioscience), IgM (II/41) (BioLegend), CD43 (S7) (BioLegend), CD24 (M1/69) (BD Biosciences), CD21 (7G6) (BD Biosciences), and BP-1/Ly-51 (6C3) (BioLegend). To identify splenic plasma cells or plasma cells from lymph node in vivo, cells positive for CD138 (281.2) (eBioscience) and negative for IgD (11-26c.2a) were considered plasma cells. For analysis of in vitro B-cell cultures, after blocking Fc receptors using anti-CD16/32 antibody, CTV-labelled cells were stained with the antibodies CD138 (281.2) and IgG1 (A85.1). For detection of vimentin using flow cytometry, anti-vimentin antibody (ERP3776) (Abcam) was used.

Tsui C., Maldonado P., Montaner B., Borroto A., Alarcon B., Bruckbauer A., Martinez-Martin N, & Batista F.D. (2018). Dynamic reorganisation of intermediate filaments coordinates early B-cell activation. Life Science Alliance, 1(5), e201800060.

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Bone Marrow Multiparameter Analysis

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Bone marrow was flushed from the femur and tibia of one leg and was used after red blood cell lysis using eBioscience RBC lysis buffer. Multi-parameter analyses of labeled cell suspensions were performed on an LSR II (Becton Dickinson) and data were analyzed with FlowJo software (TreeStar). Fluorochrome- or biotin-conjugated monoclonal antibodies (mAbs) to the following were used: mouse IA/IE (M5/114.15.2) and CD172α (P84) (both from BD Biosciences); CD11c (N418), CD209a (MMD3), CD117 (2B8), Ly6C (HK1.4), SiglecH (440c), B220 (RA3-6B2), CD135 (A2F10), CD49b (DX5) and CD19 (eBio1D3) (all from eBioscience); Ly6G (1A8) and CD3e (145-2C11); CD74 (OX6) (from Abcam); and CX3CR1 (Catalog # FAB5825P) (from RnD Systems). The streptavidin–phycoerythrin-CF594 conjugate (25-4317-82) was from BD Biosciences. The dilution of antibodies was as follows: CD117 (2B8) 1:100, CD135 (A2F10) 1:100, CD74 (OX6) 1:50, CX3CR1 (Catalog # FAB5825P) 1:50, streptavidin–phycoerythrin-CF594 conjugate (25-4317-82) 1:400, all other antibodies 1:200.

Chen J., Schlitzer A., Chakarov S., Ginhoux F, & Poidinger M. (2016). Mpath maps multi-branching single-cell trajectories revealing progenitor cell progression during development. Nature Communications, 7, 11988.

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