Mel 14

Manufactured by BD

The MEL-14 is a lab equipment product manufactured by BD. It is a device used for the measurement and analysis of various samples in a laboratory setting. The core function of the MEL-14 is to provide accurate and reliable data for research and testing purposes.

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Lab products found in correlation

Rm4 5, by BD (2 mentions) Lsrfortessa x 50 flow cytometer, by BD (1 mentions) Mp6 xt22, by BD (1 mentions) Mp6 xt22, by Thermo Fisher Scientific (1 mentions) B220 bv421 ra3 6b2, by BioLegend (1 mentions) Ly6g bv510, by BioLegend (1 mentions) Efluor 780, by BioLegend (1 mentions) Ze5 flow cytometer, by Bio-Rad (1 mentions) Clone m1 70, by BioLegend (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Facsaria, by BD (1 mentions) Facsdiva software, by BD (1 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions) Golgiplug, by BD (1 mentions) Cytofix, by BD (1 mentions) Ionomycin, by BD (1 mentions) Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions) Metformin, by Merck Group (1 mentions) Chloroquine, by Merck Group (1 mentions) Anti cd4 microbeads, by Miltenyi Biotec (1 mentions)

Close spelling variants of this product

CD62L (MEL-14; (19 citations) Anti-CD62L (MEL-14) (9 citations) CD62L (clone MEL‐14, (7 citations) Anti-CD62L (clone MEL-14, (6 citations) Anti-CD62L APC (clone mel-14; (4 citations) CD62L-FITC (MEL-14, (3 citations) Anti-CD62L APC (MEL-14, (2 citations) CD62L-PE (clone MEL-14, (2 citations) Anti-mouse CD62L (clone MEL-14) (2 citations)

9 protocols using mel 14

Multiparametric Flow Cytometry Immunophenotyping

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Antibodies were from BD Bioscience, eBioscience, and BioLegend. The following antibodies are listed in the order of antigen, fluorophore, clone, sources, catalog number, and dilution times for the final concentrations. CD11c, FITC, N418, Biolegend, 117306, 1600; CD11c, PE, HL3, BD, 553802, 800; F4/80, APC, BM8, eBioscience, 17–4801–82, 800; CD3, PerCP Cy5.5, 17A2, Biolegend, 100218, 800; B220, BV421, RA3-6B2, Biolegend, 103240, 800; B220, Al647, RA3-6B2, Biolegend, 103226, 1600; CD8a, APC-Cy7, 53-6.7, Biolegend, 100714, 800; CD8a, APC, 53-6.7, BD, 553035, 800; PD-1, BV421, 29F.1A12, Biolegend, 135221, 800; PD-L1, BV421, 10F.9G2, Biolegend, 124315, 800; IFN-gamma, PE, XMG1.2, BD, 554412, 100; IFN-gamma, FITC, XMG1.2, eBioscience, 11-7311-82, 100; TNF-alpha, FITC, MP6-XT22, BD, 560659, 100; TNF-alpha, APC, MP6-XT22, eBioscience, 560658, 100; CD62L, FITC, MEL-14, BD, 553150; CD44, PE-Cy5, IM7, BD, 561861. Antibodies were used according to the manufacturer’s instructions, including antibody dilution. Flow cytometry was conducted on a BD LSRFortessa X-50 flow cytometer at the Core Flow Cytometry Facility of Vaccine Research Center.

Zhu G., Lynn G.M., Jacobson O., Chen K., Liu Y., Zhang H., Ma Y., Zhang F., Tian R., Ni Q., Cheng S., Wang Z., Lu N., Yung B.C., Wang Z., Lang L., Fu X., Jin A., Weiss I.D., Vishwasrao H., Niu G., Shroff H., Klinman D.M., Seder R.A, & Chen X. (2017). Albumin/vaccine nanocomplexes that assemble in vivo for combination cancer immunotherapy. Nature Communications, 8, 1954.

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Neutrophil Receptor Profiling by Flow Cytometry

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Bone marrow cells were flushed with ice-cold HBSS^–++, filtered through 40 μm cell strainers, counted, pelleted at 326 × g for 10 min at 4°C, and resuspended in ice-cold DPBS⁺⁺⁺⁺ at 4 × 10⁷ cells/ml. 125 μl of cells were either kept on ice, or were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF, or with 1 μg/ml LPS, for 45 min at 37°C. Cells were sedimented at 10,000 × g for 30 s at 4°C, resuspended in Fc block (BD Biosciences, clone 2.4G2, 1:1000; for Mac1 and L-selectin) or in DPBS⁺⁺⁺⁺ (for FcγRIII), and incubated on ice for 15 min. Cells were sedimented at 10,000 × g for 30 s, resuspended in ice-cold DPBS⁺⁺⁺⁺ containing fixable viability dye (eBioscience, eFluor™ 780, 1:1000), antibodies for neutrophil markers Ly6G (Ly6G-BV510, BioLegend, clone 1A8, 1:500) and Mac1 (CD11b-AF647, BD Bioscience Clone M1/70, 1:1000), and PE-labelled antibodies for FcγRIII (CD16, BioLegend, clone S17014E, 1:100) or L-selectin (BD Biosciences clone MEL-14, 1:100), and were incubated on ice for 30 min. Cells were washed in ice-cold HBSS^–++, 1 mM EDTA, resuspended in 300 μl ice-cold HBSS^–++, 1 mM EDTA, and kept on ice. Flow cytometry was performed using a BioRad ZE5 flow cytometer, recording 20,000 neutrophils per sample. Neutrophils were identified by Ly6G^hi, CD11b^hi staining, and the mean fluorescence intensity (mfi) of receptor levels on the neutrophil surface was quantitated using FlowJo.

Hornigold K., Chu J.Y., Chetwynd S.A., Machin P.A., Crossland L., Pantarelli C., Anderson K.E., Hawkins P.T., Segonds-Pichon A., Oxley D, & Welch H.C. (2022). Age-related decline in the resistance of mice to bacterial infection and in LPS/TLR4 pathway-dependent neutrophil responses. Frontiers in Immunology, 13, 888415.

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Immunophenotyping and Cell Sorting Analysis

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Labeled antibodies specific for CD4 (RM4-5), CD44 (IM7), CD25 (PC61), CD62L (MEL14), CTLA-4/CD152 (UC10-4F10), FoxP3 (FJK-16s) and Hamster IgG1,κ (A19-3) were purchased from BD Biosciences or eBioscience. Isolated cells from lymph nodes and spleen were stained with commercially available antibodies, listed above. Intracellular staining for FoxP3 and CTLA-4 (CD152) was performed using a FoxP3 staining buffer set, according to the manufacturers’ instructions (eBioscience, San Diego, CA USA). Stained single-cell suspensions were analyzed using a BD LSRII flow cytometer running FACSDiva software (BD Biosciences, San Jose, CA USA). Cell sorting experiments were performed with a MoFlo cytometer high-speed cell sorter (Dako) or a FACSAria (BD Biosciences).

Stumpf M., Zhou X., Chikuma S, & Bluestone J.A. (2014). Tyrosine 201 of the cytoplasmic tail of CTLA-4 critically affects T regulatory cell suppressive function. European journal of immunology, 44(6), 1737-1746.

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Comprehensive Immune Cell Profiling

Cited 1 time

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Abs against mouse CD4 (RM4-5), CD3 (500A2), CD8 (53-6.7), CD62L (MEL-14), CD44 (IM7), CD25 (PC61.5), PD1 (RMP1-30), FoxP3 (FJK-16s), IFN-γ (XMG1.2), TNF-α (MP6-XT22), IL2 (JES6-5H4) were purchased from BD or eBioscience. Optimal concentrations for all fluorochrome-conjugated Abs were determined by titration. For surface staining, cells were incubated with respective Abs for 30 min at 4° C. For intra-nuclear FoxP3 staining, Fixation-Permeabilization buffer (eBioscience) was used as per manufacturer’s instruction followed by incubation with anti-FoxP3 Ab for 30 mins at 4° C. For intracellular cytokine analysis, spleen cells were incubated for 6 hours at 37° C in complete MLR media with PMA (50 ng/ml), Ionomycin (1 μg /ml) and Golgi plug (BD biosciences, for last 4 hours). After surface staining, Cytofix (BD biosciences) was used according to manufacturer’s instruction to fix and home-made perm buffer (0.1% saponin in PBS) for permeabilization followed by incubation with Abs against cytokines and analysis using flow cytometry as reported (19 (link)).

Batra L., Shrestha P., Zhao H., Woodward K.B., Togay A., Tan M., Grimany-Nuno O., Malik M.T., Coronel M.M., García A.J., Shirwan H, & Yolcu E.S. (2020). Localized Immunomodulation with PD-L1 Results in Sustained Survival and Function of Allogeneic Islets without Chronic Immunosuppression1. Journal of immunology (Baltimore, Md. : 1950), 204(10), 2840-2851.

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Murine and Human T Helper Cell Differentiation

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Naive CD4⁺ T cells (CD4⁺CD62L^hiCD44^lo) were obtained from spleens and lymph nodes of mice. Isolated naive T cells were routinely 98% pure. CD4⁺ T cells were purified from spleen and lymph nodes with anti-CD4 microbeads (Miltenyi Biotec), and then were further sorted as naive CD4⁺CD62L^hi T cells by flow cytometry with antibodies anti-CD4-FITC (RM4-4, BD Biosciences) and anti-CD62L-Alexa 700 (MEL-14, BD Biosciences). Isolated naive CD4⁺ T cells were stimulated with plate-bound antibodies against CD3 (145-2C11, 2 μg ml⁻¹, BioXcell) and CD28 (PV-1, 2 μg ml⁻¹, BioXcell) and polarized into effector CD4⁺ T lymphocyte subsets without cytokines (T_H0 cells), or with IL-12 (20 ng ml⁻¹) for T_H1 cells, or with IL-4 (20 ng ml⁻¹) for T_H2 cells, or with TGF-β (2 ng ml⁻¹) and IL-6 (25 ng ml⁻¹) for T_H17 cells, or with mouse TGF-β (2 ng ml⁻¹) and IL-4 (20 ng ml⁻¹) for T_H9 cells. In some experiments, metformin or chloroquine were added (Sigma-Aldrich, France). Cells were classically harvested on day 3 (unless otherwise specified) for detection of cytokines by ELISA and RT-qPCR analysis. For human in vitro T-cell differentiation, naive T cells were sorted from healthy donors and stimulated with plate-bound antibodies against CD3 (5 μg ml⁻¹, BioXcell) and CD28 (5 μg ml⁻¹, BioLegend) and polarized into T_H9 cells with TGF-β (10 ng ml⁻¹) and IL-4 (5 ng ml⁻¹) (R&D systems).

Rivera Vargas T., Cai Z., Shen Y., Dosset M., Benoit-Lizon I., Martin T., Roussey A., Flavell R.A., Ghiringhelli F, & Apetoh L. (2017). Selective degradation of PU.1 during autophagy represses the differentiation and antitumour activity of TH9 cells. Nature Communications, 8, 559.

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Antigen-Specific T Cell Staining Assay

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For antigen-specific T cells staining, PE-conjugated (K^b-IMYNYPAM)₄ tetramer (PE EsxH TET), or (H-2K^b)₄-SIINFEKL (PE OVA TET) (MBL International, MA, USA) tetramers were used. Tetramer staining of whole blood was performed with TET at room temperature for 10 min before adding the other surface markers: PerCP-Vio700-anti-CD3 (REA606, Miltenyi) and FITC-anti-CD8α (KT15, Santa Cruz) mAbs at 4 °C for an additional 20 min. Tetramer staining of the T splenocytes was performed with TET at room temperature for 10 min before further staining with BV650-anti-CD3 (145-2C11, BD Biosciences), FITC-anti-CD8a (KT15, Santa Cruz), PE-Cy5-anti-CD127 (A7R34, BD Biosciences), PE-Dazzle-anti-KLRG1 (2F1, Biolegend), AF700-anti-CD62L (MEL-14, BD Biosciences), and APC-Cy7-anti-CD44 (IM7, BD Biosciences) mAbs at 4 °C for an additional 20 min incubation. Prior to surface staining, cells were treated with 2.4G2 to block Fc receptors. Cells were washed twice before acquisition.

Ku M.W., Authié P., Nevo F., Souque P., Bourgine M., Romano M., Charneau P, & Majlessi L. (2021). Lentiviral vector induces high-quality memory T cells via dendritic cells transduction. Communications Biology, 4, 713.

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Multi-Marker Immune Profiling of T Cells

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Anti-mouse: α-CD45 (30-F11, BD, 4 μg ml⁻¹), α-CD3 (145-2C11, Biolegend, 5 μg ml⁻¹), α-CD4 (GK1.5, Biolegend, 5 μg ml⁻¹), α-CD8 (53-6.7, BD, 5 μg ml⁻¹), α-CTLA-4 (1B8, abcam, 30 μg ml⁻¹), α-TIM3 (B8.2C12, Biolegend, 2.5 μg ml⁻¹), α-OX40 (OX-86, Biolegend, 10 μg ml⁻¹), α-CD62L (MEL-14, BD, 5 μg ml⁻¹), α-CD44 (IM7, Biolegend, 2.5 μg ml⁻¹), α-LAG-3 (C9B7W, Biolegend, 5 μg ml⁻¹), α-IFN-γ (XMG1.2, Biolegend, 2.5 μg ml⁻¹), α-TNF-α (MP6-XT22, Biolegend, 2.5 μg ml⁻¹), α-Granzyme B (NGZB, ThermoFisher, 1.25 μg ml⁻¹), α-FOXP3 (MF-14, Biolegend, 10 μg ml⁻¹), α-Ki67 (SolA15, ThermoFisher, 0.6 μg ml⁻¹). Anti-human: α-CD4 (A161A1, Biolegend, 2.5 μg ml⁻¹), α-T-bet (4B10, Biolegend, 2.5 μg ml⁻¹), α-Phospho-Akt (Ser473) (D9E, Cell Signaling, 0.5 μg ml⁻¹), α-Phospho-Akt (Thr308) (D25E6, Cell Signaling, 0.5 μg ml⁻¹), α-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E, Cell Signaling, 0.5 μg ml⁻¹).

Ho W.S., Wang H., Maggio D., Kovach J.S., Zhang Q., Song Q., Marincola F.M., Heiss J.D., Gilbert M.R., Lu R, & Zhuang Z. (2018). Pharmacologic inhibition of protein phosphatase-2A achieves durable immune-mediated antitumor activity when combined with PD-1 blockade. Nature Communications, 9, 2126.

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Characterization of B Cell Subsets

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Blood samples were collected from the superficial temporal vein. Spleens were mechanically separated and digested in 1 mg/ml Collagenase II for 45 min at 37°C. For both blood and spleen samples, RBCs were lysed (Pharm Lyse, BD Biosciences) prior to counting (Cellometer X4, Nexcelom Bioscience) and processed for flow cytometeric staining. Samples were FcR-blocked (2.4G2; BioLegend), then stained on ice with specific Abs; CD45 Pacific Blue conjugated (30-Fl1; BioLegend), CD19 PE conjugated (eBio1D3; eBioscience), B220 PE conjugated (RA3-6B2; eBioscience), MHC II allophycocyanin/Cy7 conjugated (I-A/I-E) (M5/114; eBioscience), GL7 allophycocyanin conjugated (GL7; BioLegend), CD4 allophycocyanin conjugated (CK1.5; eBioscience), CD8α PerCP conjugated (53-6.7; BD Pharmingen), CD23 Fitc conjugated (B3B4; BioLegend), CD21 PEcy7 conjugated (7E9; BioLegend), TCRβ Fitc conjugated (H57-597; eBioscience), PD1 PE conjugated (29F.1A12; BioLegend), CXCR5 PerCPcy5.5 conjugated (L138D7; BioLegend), CD44 allophycocyanin/Cy7 conjugated (IM7; BioLegend), and CD62L PEcy7 conjugated (MEL-14; BD Biosciences). Samples were run on BD LSRFortessa instrument and analyzed using FlowJo 10 (Tree Star). B cell subsets were identified as follicular (CD19⁺ CD21⁻ CD23⁺), MZ (CD19⁺ CD21⁺ CD23⁻), or germinal center (GC) (CD19⁺ GL7⁺).

Williams J.W., Elvington A., Kessler S., Wohltmann M., Wu G.F, & Randolph G.J. (2019). B Cell–Mediated Antigen Presentation through MHC Class II Is Dispensable for Atherosclerosis Progression. ImmunoHorizons, 3(1), 37-44.

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T-Lymphocyte Immunophenotyping by Flow Cytometry

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After culture, T-lymphocytes were counted and resuspended in pre-warmed fresh phenol red-free culture media (see aforementioned media recipe). Cells were blocked using anti-CD16/CD32 antibody (BD Biosciences clone 2.4G2, #553141, San Jose, CA) prior to staining. The following antibodies were used at 1:100 to identify naïve, effector, or memory lineages of T-lymphocytes: APC-CD44 (BD Biosciences clone IM7, # 561862, San Jose, CA) and BV421-CD62L (BD Biosciences clone MEL-14, # 562910, San Jose, CA). Antibodies were added and incubated for 30 min at 37°C, and then washed twice using pre-warmed fresh media. T-lymphocytes were immediately analyzed by flow cytometry on a LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and quantified using FlowJo cytometric analysis software (Tree Star, Ashland, OR). Cell lineages were defined as follows: Naïve, CD62L+, CD44-; Effector/Effector Memory, CD62L-, CD44+; Central Memory, CD62L+, CD44+. Immunophenotyping was performed in warmed media due to concurrent superoxide measurements (see below).

Case A.J., Roessner C.T., Tian J, & Zimmerman M.C. (2016). Mitochondrial Superoxide Signaling Contributes to Norepinephrine-Mediated T-Lymphocyte Cytokine Profiles. PLoS ONE, 11(10), e0164609.

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