Melanoma 624 wt or knockout cells were plated at equal densities and incubated overnight. Resuspended cells were incubated on ice for 1 h with the primary antibody at a concentration of 0.2 μg/well in FACS buffer (1× PBS, 0.5% bovine serum albumin, 0.05% NaN3). The cells were then incubated for 30 min on ice with anti-mouse AlexaFluor 647 secondary antibody (Jackson ImmunoResearch). The following primary antibodies were used: anti-MICA (clone 159227, R&D Systems), anti-MICB (clone 236511, R&D Systems), anti-ULBP1 (clone 170818, R&D Systems), anti-ULBP2/5/6 (clone 165903, R&D Systems), anti-ULBP3 (clone 166514, R&D Systems), anti-B7H6 (clone 875001, R&D systems), anti-PVR (in-house developed), anti-HLA1 (W6/32), anti-Beta-2 microglobulin (β2M, clone 2M2, Biolegend), anti-Ceacam-1 (clone ASL-32, Biolegend), anti-Nectin-2 (clone TX31, Biolegend). Mouse IgG1 (clone MOPC-21, Biolegend), IgG2a (clone MOPC-173, Biolegend), and IgG2b (clone MPC-11, Biolegend) were used as an isotype control.
Mouse igg1 clone mopc 21
Manufactured by BioLegend
Sourced in United States, Germany
Mouse IgG1 (clone MOPC-21) is an antibody isotype control that can be used to determine the background signal in immunoassays and flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti mica clone 159227, by R&D Systems (1 mentions)
Anti ulbp3, by R&D Systems (1 mentions)
Anti micb clone 236511, by R&D Systems (1 mentions)
Anti ceacam 1 clone asl 32, by BioLegend (1 mentions)
Igg2a clone mopc 173, by BioLegend (1 mentions)
Fc receptor blocking reagent, by Miltenyi Biotec (1 mentions)
Clone md 1, by BioLegend (1 mentions)
Fitc conjugated anti human hla dr and dp clone hl 38, by Immunotools (1 mentions)
Fitc conjugated mouse igg2a, by Immunotools (1 mentions)
Anti tlr2 antibody, by BioLegend (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Properdin, by Quidel (1 mentions)
Hrp conjugated goat anti mouse, by Agilent Technologies (1 mentions)
C3a eia kit, by Quidel (1 mentions)
Bdp 13176, by MedChemExpress (1 mentions)
Dimethyl sulfoxide (dmso), by Carl Roth (1 mentions)
Mouse igg2b clone mpc 11, by BioLegend (1 mentions)
Alexa fluor 647 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Cytoflex cytometer, by Beckman Coulter (1 mentions)
7 protocols using mouse igg1 clone mopc 21
1
Flow Cytometry Immunophenotyping of Melanoma Cells
2
Integrin-mediated Adhesion of MDMs and MDDCs
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The role of CD11b/CD18 and CD11c/CD18 in the adhesion to fibrinogen was analyzed by comparing the adhesive properties of MDMs and MDDCs treated with either 50 μg/ml anti-CD11b antibody (monoclonal mIgG1 clone ICRF44, Biolegend) or 50 μg/ml anti-CD11c antibody (monoclonal mIgG1 clone 3.9, Biolegend). Both antibodies are specific for the ligand binding domain of the corresponding integrin and were used in sterile, azide-free form at saturating concentration previously titrated by flow cytometry. The effect of receptor specific antibodies was compared to samples incubated with an isotype matched control antibody (mouse IgG1 clone MOPC-21, Biolegend). To exclude Fc-receptor mediated binding of the blocking monoclonal antibodies we used an Fc-receptor blocking reagent (Miltenyi Biotech) prior to the antibody treatment. Cells were incubated with the receptor-specific antibodies for 30 minutes at 4°C then used in the functional studies without washing, since unblocked integrins are known to recycle and appear on the cell surface from an intracellular pool [35 (link)], that would decrease the efficiency of receptor blocking.
3
Monoclonal Antibodies for IFN-γ Neutralization Assays
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For ELISA and functional assays, monoclonal antibodies against the IFN-γ clones B27 and B133.5 were obtained from ImmunoTools in Germany, and clone MD-1 was purchased from BioLegend Inc. in CA. These antibodies were IgG1-specific to IFN-γ and exhibited neutralizing capacity against IFN-γ (10 (link)–12 (link)). For the neutralization assay, mouse IgG1 (clone MOPC-21) (BioLegend Inc.) was used as the isotype-matched antibody control. For flow cytometry, FITC-conjugated anti-human HLA-DR and DP (clone HL-38) and isotype-matched control FITC-conjugated mouse IgG2a, were purchased from ImmunoTools in Germany. PE-conjugated anti-human phospho-STAT1 (pY701) antibody (BD Pharmingen) was purchased from BD Biosciences. Recombinant human IFN-γ (rIFN-γ) was purchased from R&D Systems in MN.
4
TLR2 Modulation of Murine PP Cell Response
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Murine PP cells were preincubated with 0.5 µg/ml anti-TLR2 antibody (purified anti-mouse/human CD282, clone T2.5, Biolegend, San Diego, CA, USA) or isotype antibody (purified mouse
IgG1, clone MOPC-21, Biolegend) at 37°C for 30 min and then cultured with or without the purified MVs (Fr. 3 obtained by the density gradient ultracentrifugation) or L.
sakei NBRC15893 (50 µg/well) cells for 4 days at 37°C under 5% CO2 in air.
IgG1, clone MOPC-21, Biolegend) at 37°C for 30 min and then cultured with or without the purified MVs (Fr. 3 obtained by the density gradient ultracentrifugation) or L.
sakei NBRC15893 (50 µg/well) cells for 4 days at 37°C under 5% CO2 in air.
5
Complement pathway protein detection
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Complement proteins FH (#341274), FB (#341262), C3b (#204860), C3 (#204885), factor I (FI) (#341280), C1q (#204876), goat anti-FB (#341272) and goat anti-FH (#341276) antiserum were obtained from Merck (Darmstadt, Germany; obtained via Merck Life Science Kft., Budapest, Hungary). FD (#A409), properdin (#A412), mouse anti-C5b-9 (#A239), anti-FB (Bb) (#A227), anti-FB (Ba) (#A225) and C3a EIA kit (#A032) were from Quidel (obtained via Biomedica, Budapest, Hungary). Purified human IgG (#I2511), human serum albumin (#A3782), human alpha-1 antitrypsin (#SRP6312), antibodies against C1q (#234390), human IgG (#A6029), IgM (#A0420), IgA (#A0295), IgG1 (clone HP-6001, #I9388), IgG2 (clone HP-6002, #I9513), IgG3 (clone HP-6050, #I7260), IgG4 (clone HP-6025, #I7385), IgGκ (clone KP-53, #K4377), IgGλ (clone HP-6054, #L6522), and avidin-HRP (#A7419) were purchased from Merck. Mouse IgG1 (clone MOPC-21, #BZ-400101) was obtained from BioLegend (San Diego, CA). MBL (#9086-MB) and biotin-conjugated goat anti-MBL (#BAF2307) were from R&D Systems (Minneapolis, MN), HRP-conjugated goat anti-mouse (#P0447), rabbit anti-goat (#P0449) and swine anti-rabbit (#P0217) antibodies were from DAKO (Hamburg, Germany). Goat anti-C3 F(ab’)2 (#55062) and HRP-conjugated goat anti-C3 F(ab’)2 (#55237) were from MP Biomedicals (Solon, OH).
6
Fscn1 Inhibitor Effects on Immune Cells
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Fscn1 inhibitors NP-G2-044 (Selleckchem, Houston, TX, USA) and BDP-13176 (MedChemExpress, Monmouth Junction, NJ, USA) were reconstituted in DMSO (Roth, Karlsruhe, Germany). APC-eFl70-labeled anti-CD11c (clone N418), FITC-MHCI (28148), eFl450-MHCII (M5/114.15.2), APC-CD40 (1C10), PerCP-eFl710-CD80 (16-10A1), PE/Cy7-CD86 (GL-1), PE/TexasRed-CD274/PD-L1 (10F.9G2), PE-CD273/PD-L2 (Ty25), eFl506-CD3 (145-2C11), SB600-CD11b (M1/70), SB702-CD19 (eBio103), PE-NK1.1 (PK136), PE-eFl610-Ly6G (1A8-L6g), eFl780-FVD and eFl450-FVD used for flow cytometric analysis were purchased from BD Biosciences (Franklin Lakes, NJ, USA), BioLegend (San Diego, CA, USA) or ThermoFisher (Waltham, MA, USA). Unlabeled mouse anti-human Fscn1 antibody (clone 55K2; Sigma-Aldrich, Deisenhofen, Germany), a corresponding isotype control antibody (mouse IgG1, clone MOPC-21, BioLegend), secondary AF488-labeled IgG goat anti-mouse antibody (948492), AF647-labeled anti-CD11c antibody (clone N418), Hoechst (nuclear staining) and Alexa Fluor 555 phalloidin (F-actin) (all from ThermoFisher) were used for confocal laser scanning analysis (CLSM).
7
Flow Cytometry Analysis of CLL B Cells
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We carried out flow cytometry analyses directly after cell isolation and on the 3rd day of cell culture. To characterize CLL B cells the following antibodies were used: anti-CD11b (clone ICRF44, IgG1, Biolegend, San Diego, CA, USA), anti-CD11c (clone BU15, IgG1, ImmunoTools GmbH, Friesoythe, Germany), anti-CD41a (clone HIP8, IgG1, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), anti-CD51/CD61 (clone 23C6, IgG1, Invitrogen) anti-CD49e (clone SAM1, IgG2b, Invitrogen) with Alexa Fluor 647-conjugated goat anti-mouse antibodies (Thermo Fisher Scientific Inc.). As isotype control mouse IgG1 (clone MOPC-21, Biolegend) and mouse IgG2b (clone MPC-11, Biolegend) were used. To rule out dead cells from the analyses we used propidium iodid (Thermo Fisher Scien-tific Inc.) staining. Measurement was performed on a CytoFLEX cytometer (Beckman Coulter Life Sciences, Indianapolis, IN) using the CyteExpert software. Data were analysed using the CytExpert and Kaluza softwares.
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