Cd138 clone 281 2

Manufactured by BD

Sourced in Australia

CD138 (clone 281-2) is a monoclonal antibody used for the identification and detection of CD138 antigen. CD138, also known as Syndecan-1, is a cell surface proteoglycan expressed on plasma cells and some epithelial cells. This antibody can be used in various laboratory applications, such as flow cytometry and immunohistochemistry, to study the expression and distribution of CD138 in biological samples.

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CD138 (281-2, (6 citations) Biotinylated anti-mouse CD138 (clone 281-2; (2 citations) CD138-APC (clone 281–2; (2 citations) Anti-CD138 (clone 281–2, (2 citations) APC-conjugated anti-CD138 (clone 281-2) (2 citations)

8 protocols using cd138 clone 281 2

Comprehensive Immune Cell Analysis

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Spleens and cLNs were harvested and dissociated by mechanical dispersion. Following RBC lysis with ACK Lysis Buffer (Lonza), cells were incubated with Fc block (CD16/32, clone 2.4G2, BD Biosciences) and treated with antibodies directed against the following as indicated: B220 (clone RA3-6B2, BD Biosciences), CD23 (clone B3B4, Biolegend), CD21/35 (clone 7G6, BD Biosciences), T-bet (clone 4B10, BD Biosciences), CD11c (clone HL3, BD Biosciences), CD4 (clone GK1.5, BD Biosciences), CD8α (clone 53-6.7, BD Biosciences), CD44 (clone IM7, BD Biosciences), CD62L (clone MEL-14, BD Biosciences), CD138 (clone 281-2, BD Biosciences), CD69 (clone H1.2F3, Biolegend), and TLR7 (clone A94B10, BD Biosciences). Data were acquired using a BD Biosciences Fortessa and analyzed with FlowJo software (BD Biosciences).

Punnanitinont A., Kasperek E.M., Kiripolsky J., Zhu C., Miecznikowski J.C, & Kramer J.M. (2022). TLR7 agonism accelerates disease in a mouse model of primary Sjögren’s syndrome and drives expansion of T-bet+ B cells. Frontiers in Immunology, 13, 1034336.

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Multiparameter Flow Cytometry Isolation

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Flow cytometry was performed as previously described [9 (link)]. Spleen and cervical lymph nodes (cLNs) were mechanically dissociated and treated with RBC lysis buffer. Cells were incubated with Fc block (Cd16/32, clone 2.4G2, BD Biosciences) and were treated with the following antibodies as indicated: B220 (clone RA3-6B2, BD Biosciences), CD23 (clone B3B4, BioLegend), CD21/35 (clone 7G6, BD Biosciences), CD4 (clone GK1.5, BD Biosciences), CD69 (clone H1.2F3, BioLegend), CD86 (Clone GL1, BD Biosciences), CD11c (clone HL3, BD Biosciences), CD11b (clone M1/70, BD Biosciences), and CD138 (Clone 281-2, BD Biosciences). Data were acquired using a Fortessa (BD Biosciences) and analyzed using FlowJo software (BD Biosciences).
Sort-purification was performed using a FACSAria (BD Biosciences). Splenocytes were incubated with antibodies directed against B220, CD4, CD11b and CD11c as described above. B cells (B220+), T cells (CD4+) or monocytes (CD11b+, CD11c+, and CD11b/CD11c double positive) (1 × 10⁵ cells) were sorted directly into lysis buffer for RNA isolation (RNeasy Plus Mini Kit, Qiagen). For stimulation experiments, B220+ cells were sorted into media and cultured overnight with either culture media alone or culture medium containing either LPS or anti-IgM/IgG + IL-4, as described above.

Kiripolsky J., Kasperek E.M., Zhu C., Li Q.Z., Wang J., Yu G, & Kramer J.M. (2021). Tissue-specific activation of Myd88-dependent pathways governs disease severity in primary Sjögren’s syndrome. Journal of autoimmunity, 118, 102608.

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Comprehensive B Cell Immunophenotyping

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For flow cytometry, we used fluorochrome- or biotin-labeled antibodies against B220 (clone RA3–6B2), CD1d (clone 1B1), CD16/CD46 (Fc block; clone 2.4G2), CD19 (clone 1D3), CD21/CD35 (clone 7G6), CD23 (clone B3B4), CD35 (clone 8C12), CD38 (clone 90), CD95 (clone Jo2), CD138 (clone 281-2), GL-7 (clone GL-7), IgD (clone 11–26c.2a), IgM (clone R6–60.2), IgM^a (clone DS-1), IgM^b (clone AF6-78), and C3 split products (clone RmC11H9); all were purchased from BD Biosciences, Biolegend, eBioscience, or Cedarlane Laboratories. Rabbit polyclonal IgG anti-SRBC was prepared in-house (26 (link)). Staining was done in FACS buffer (PBS 2% FBS; Sigma Aldrich) or, when two or more Brilliant Violet™ antibody conjugates were used, in Brilliant Stain Buffer (BD Biosciences). All antibodies were used at ≤0.5 µg per 1x10⁶ cells.

Palm A.K., Westin A., Ayranci D, & Heyman B. (2024). Endogenous complement-activating IgM is not required for primary antibody responses but promotes plasma cell differentiation and secondary antibody responses to a large particulate antigen in mice. Frontiers in Immunology, 14, 1323969.

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Analyzing Germinal Center Responses to Adjuvanted Vaccines

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Mice were s.c. immunized with alum-adsorbed NP–OVA (50 µg) and TLR7–alum or TLR7-NP (equivalent gardiquimod dose, 20 µg) in 100 µl PBS at tail base. Inguinal dLNs were excised at day 4, day 7, day 14 and day 22 to prepare single-cell suspensions. For flow cytometry analysis of GC B cells, follicular T cells (TFH) and plasmablasts, cells from the dLNs were stained with Ghost Dye Violet 510 (Tonbo Biosciences). Cells were then washed and blocked with Fc-blocker (clone 2.4G2, BD Bioscience) prior to staining with markers, including CD19 (clone 1D3/CD19, Biolegend), CD38 (clone 90, BD Biosciences), CD95 (clone Jo2, BD Biosciences), CD138 (clone 281-2, BD Biosciences), CD44 (clone IM7, BioLegend), CD3 (clone 17A2, BioLegend), CD4 (clone GK1.5, BioLegend), CXCR5 (clone L138D7, BioLegend) and PD1 (clone 29F.1A12, BioLegend). After staining, cells were washed and fixed with 1.5% PFA. Stained cells were collected using BD FACS Diva v8.01 software associated with a BD LSRII flow cytometer. Data were analysed with FlowJo 10 software. The gating strategy consisted of gating GCBC on live single CD3⁻CD19⁺CD95⁺CD38⁻ cells, TFH on live single CD19⁻CD3⁺CD4⁺CXCR5⁺PD1^hi, and plasmablasts on live single CD138⁺CD44⁺ cells.

Yin Q., Luo W., Mallajosyula V., Bo Y., Guo J., Xie J., Sun M., Verma R., Li C., Constantz C.M., Wagar L.E., Li J., Sola E., Gupta N., Wang C., Kask O., Chen X., Yuan X., Wu N.C., Rao J., Chien Y.H., Cheng J., Pulendran B, & Davis M.M. (2023). A TLR7-nanoparticle adjuvant promotes a broad immune response against heterologous strains of influenza and SARS-CoV-2. Nature Materials, 22(3), 380-390.

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Immune Cell Analysis in Virus-Infected Mice

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Mice infected with virus for 10 days were anesthetized and perfused. Mouse spleens were harvested and homogenized. Splenocytes were treated twice with buffer to lyse red blood cells and washed. In addition, leukocytes in the brain were separated by discontinuous Percoll gradient and washed. Cells were stained with FITC- or PE-conjugated control antibodies or monoclonal antibodies against CD4 (clone GK 1.5, eBioscience), CD8a (clone 53–6.7, eBioscience), CD19 (clone 6D5, eBioscience), CD69 (clone H1.2F3, eBioscience), or CD138 (clone 281–2, BD Biosciences). The stained cells were analyzed by a FACSCalibur (BD Biosciences) and data were analyzed by WinMDI software.

Wang L.C., Yao H.W., Chang C.F., Wang S.W., Wang S.M, & Chen S.H. (2017). Suppression of interleukin-6 increases enterovirus A71 lethality in mice. Journal of Biomedical Science, 24, 94.

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Comprehensive B Cell Immunophenotyping

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Bone marrow and spleen single cell suspensions were stained for dead cell exclusion using Live/Dead fixable violet dead cell staining kit (Invitrogen Life Technologies, Grand Island, NY). Surface markers were stained with a mixture of fluorochrome-conjugated antibodies, which included: CD19 (clone 6D5, BioLegend, San Diego, CA), B220 (clone RA3-6B2, eBioscience, San Diego, CA), CD138 (clone 281–2, BD bioscience, San Jose, CA), IgD (clone 11.26c.2a, BioLegend, San Diego, CA), IgM (clone II/41, eBioscience, San Diego, CA), IgG (eBioscience, San Diego, CA), MHC class II (clone 500A2, eBioscience, San Diego, CA), CD80 (clone I6-10A1, BD bioscience, San Jose, CA) CD86 (clone GL-1, BioLegend, San Diego, CA).
Fluorescence minus one (FMO) controls were included in each staining protocol and used to set specific gates (22 (link), 26 , 27 (link)). 6-peak validation beads were used for calibration during the time course analysis. Samples were run on a 12-color LSRII cytometer (BD bioscience, San Jose, CA) and analyzed by Flow Jo software (Tree Star Inc., Ashland, OR).

Ramon S., Baker S.F., Sahler J.M., Kim N., Feldsott E.A., Serhan C.N., Martínez-Sobrido L., Topham D.J, & Phipps R.P. (2014). The specialized proresolving mediator 17-HDHA enhances the antibody-mediated immune response against influenza virus: Anew class of adjuvant?. Journal of immunology (Baltimore, Md. : 1950), 193(12), 6031-6040.

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Determination of Absolute Live Cell Number

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Absolute cell number was determined with addition of 1x10 4 calibration beads directly to cells prior to analysis. 0.2mM Propidium iodide (PI) was also added with the beads to identify dead cells by exclusion. Ratio of live cells to beads was measured by flow-cytometry to determine the absolute live cell number in culture. Cohort analysis was performed as described by Hawkins et al. (2007a) For in vitro differentiation studies, B cells were stained using CD138 (clone 281-2, Cat#564068, BD PharMingen).
In vitro drug preparation JQ1 (a kind gift from Prof. Ricky Johnstone -Peter MacCallum Cancer Centre, Parkville, Victoria, Australia) and PRMT5 inhibitor (see the synthesis of CTX-0391034 section below) were diluted in dimethyl sulfoxide (DMSO) and used at concentrations indicated in figures.

Kong I.Y., Rimes J.S., Light A., Todorovski I., Jones S., Morand E., Knight D.A., Bergman Y.E., Hogg S.J., Falk H., Monahan B.J., Stupple P.A., Street I.P., Heinzel S., Bouillet P., Johnstone R.W., Hodgkin P.D., Vervoort S.J, & Hawkins E.D. (2020). Temporal Analysis of Brd4 Displacement in the Control of B Cell Survival, Proliferation, and Differentiation. Cell reports, 33(3).

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Multiparameter Flow Cytometry Panel

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The following antibodies and staining reagents were used: IgG1 (clone A85-1), IgG2a/b (clone R2-40), CD138 (clone 281-2), IgM (clone 11–41), CD24 (clone M1/69), CD4 (clone RM4-5), CD21/35 (clone 7E9), CD8 (clone 53-6.7), CD25 (clone 3C7), CD62L (clone MEL-14), CD69 (clone H1.2F3), CD44 (clone lM7), CD11c (clone HL3), CD80 (clone 16-10A1), CD86 (clone GL1), CD11b (clone M1/70), c-Kit (CD117, clone 2B8), streptavidin-PerCP, from BD Pharmingen; Plvap (clone MECA-32) was from Abcam; CD38 (clone 90), B220 (clone 6B2), CD23 (clone B3B4), and IgD (clone 11-26c), FceRI (clone MAR-1) from eBioscience; peanut agglutinin from Vector Laboratories. MECA-32 mAb (anti mouse Plvap rat IgG2a) secreting hybridoma was obtained from the Developmental Studies Hybridoma Bank (DSHB, U. Iowa) was produced in serum free conditions by BioXCell (Lebanon, NH). The antibody was labeled with AlexaFluor (AF) fluorochromes using the protein labeling kits for AF488, and AF647 (Invitrogen), as per manufacturer’s instructions. Flow cytometry was performed on either a refurbished FACSCAN or a FACSCalibur running CellQuest software (BD), or a FACSCanto running FACSDiva software within the Norris Cotton Cancer Center DartLab Immune Monitoring Facility. The data analysis performed using FlowJo (Tree Star, Inc.).

Elgueta R., Tse D., Deharvengt S.J., Luciano M.R., Carriere C., Noelle R.J, & Stan R.V. (2016). Endothelial Plasmalemma Vesicle Associated Protein regulates the homeostasis of splenic immature B cell and B1 B cells. Journal of immunology (Baltimore, Md. : 1950), 197(10), 3970-3981.

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