fitc goat anti mouse ig, by BD - Product details

1

Immunophenotyping of Neuroblastoma Cells

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Single-cell suspensions of approximately 1×10⁶ elements were treated with 10% FcR blocking reagent (130-059-901, Miltenyi Biotec Inc. Auburn, CA) for 10 min at 4°C and then stained with anti-human nectin-1 (R1.302.12) (sc-69718, Santa Cruz Biotechnology, Inc. Dallas, TX), nectin-2 (R2.525) (sc-32804, Santa Cruz Biotechnology, Inc.) or HVEM (CW10) (sc-21718, Santa Cruz Biotechnology, Inc.). After applying the unconjugated primary Abs or their isotype controls, cells were stained with a secondary FITC-conjugated goat anti-mouse IgG (554001, BD Biosciences, San Jose, CA). For 3-O-Sulfated Heparan Sulfate (3-OS HS) analysis, cells were first fixed in 1% paraformaldehyde (PFA) for 10 min at 4°C followed by incubation with antibody HS4C3 (30 (link)) for 1 hour at 4°C. Next, cells were stained with unconjugated mouse anti-VSV (P5D4) (V5507, Sigma-Aldrich Corp. St. Louis, MO) for 30 min at 4°C. Finally, cells were stained with FITC goat anti-mouse Ig (554001, BD Biosciences) for 20 mins at 4°C. The analysis has been performed on the neuroblastoma panel for three independent times. Average mean fluorescence intensities (MFI) of nectin-1, nectin-2, HVEM and 3-OS HS relative to isotype (for netin-1, nectin-2 and HVEM) or unstained controls (for 3-OS HS) were calculated and presented as a heatmap in Figure 3c.

Wang P.Y., Swain H.M., Kunkler A.L., Chen C.Y., Hutzen B.J., Arnold M.A., Streby K.A., Collins M.H., Dipasquale B., Stanek J.R., Conner J., van Kuppevelt T.H., Glorioso J.C., Grandi P, & Cripe T.P. (2015). Neuroblastomas Vary Widely in Their Sensitivities to Herpes Simplex Virotherapy Unrelated to Virus Receptors and Susceptibility. Gene therapy, 23(2), 135-143.

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2

Immunohistochemical Detection of Adrenergic Receptors and P-gp in Cancer Cells

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Cells were seeded into glass Lab-Tek Chamber Slides (8 wells; 0.8cm2/well) at a density of 15–20 × 10³/well, allowed to attach for 24 h at 37 °C. For P-gp detection AS cells were previously treated with 1 µg/ml docetaxel, 150 µM propranolol and their combination. Once the confluence was reached the cells were fixed for 10 min in acetone at RT and treated with a blocking solution. Then, the slides were incubated with diluted primary antibody anti-β1 Adrenergic Receptor (Abcam), anti-β2 Adrenergic Receptor (Abcam), anti-β3 Adrenergic Receptor (Abcam) and anti-P-gp (JSB-1, Abcam) in 1% BSA in PBS for 1 h at RT. After 3 wash steps with PBS, cells were then incubated with EnVision + System-HRP Labelled polymer secondary antibody (Dako, Denmark). Color development was obtained with AEC ready to use solution (Dako, Denmark), whereas the nuclei were counterstained with hematoxylin. Then, the slides were sealed with Kaiser mounting medium for optical observation.
For the evaluation of CD-31 and CD-34,the slides were incubated with diluted primary antibody anti-CD-31 (PECAM-1, Novocastra) and anti-CD-34 (clone QBEND-10, Novocastra) in 3% BSA in PBS for 1 h and revealed by secondary antibody FITC-Goat anti-mouse Ig (BD Biosciences). Thereafter the slides were sealed with Vectashield (Vector Laboratories) for fluorescence examination by means of DMi8 Leica microscope.

Porcelli L., Garofoli M., Di Fonte R., Fucci L., Volpicella M., Strippoli S., Guida M, & Azzariti A. (2020). The β-adrenergic receptor antagonist propranolol offsets resistance mechanisms to chemotherapeutics in diverse sarcoma subtypes: a pilot study. Scientific Reports, 10, 10465.

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3

Immunophenotyping of Neuroblastoma Cells

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Single-cell suspensions of approximately 1×10⁶ elements were treated with 10% FcR blocking reagent (130-059-901, Miltenyi Biotec Inc. Auburn, CA) for 10 min at 4°C and then stained with anti-human nectin-1 (R1.302.12) (sc-69718, Santa Cruz Biotechnology, Inc. Dallas, TX), nectin-2 (R2.525) (sc-32804, Santa Cruz Biotechnology, Inc.) or HVEM (CW10) (sc-21718, Santa Cruz Biotechnology, Inc.). After applying the unconjugated primary Abs or their isotype controls, cells were stained with a secondary FITC-conjugated goat anti-mouse IgG (554001, BD Biosciences, San Jose, CA). For 3-O-Sulfated Heparan Sulfate (3-OS HS) analysis, cells were first fixed in 1% paraformaldehyde (PFA) for 10 min at 4°C followed by incubation with antibody HS4C3 (30 (link)) for 1 hour at 4°C. Next, cells were stained with unconjugated mouse anti-VSV (P5D4) (V5507, Sigma-Aldrich Corp. St. Louis, MO) for 30 min at 4°C. Finally, cells were stained with FITC goat anti-mouse Ig (554001, BD Biosciences) for 20 mins at 4°C. The analysis has been performed on the neuroblastoma panel for three independent times. Average mean fluorescence intensities (MFI) of nectin-1, nectin-2, HVEM and 3-OS HS relative to isotype (for netin-1, nectin-2 and HVEM) or unstained controls (for 3-OS HS) were calculated and presented as a heatmap in Figure 3c.

Wang P.Y., Swain H.M., Kunkler A.L., Chen C.Y., Hutzen B.J., Arnold M.A., Streby K.A., Collins M.H., Dipasquale B., Stanek J.R., Conner J., van Kuppevelt T.H., Glorioso J.C., Grandi P, & Cripe T.P. (2015). Neuroblastomas Vary Widely in Their Sensitivities to Herpes Simplex Virotherapy Unrelated to Virus Receptors and Susceptibility. Gene therapy, 23(2), 135-143.

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4

Flow Cytometric Analysis of PBMCs

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PBMC were analyzed using a 3-laser BD LSRII flow cytometer and FlowJo software (Treestar) as described (9 (link)). Primary antibodies were anti-Apc1 (Novus; NBP1-77375), anti-PGK1 (Abcam; ab67335), anti-γH2AX (Cell Signaling; #9718), and anti-phospho-NF-κB p65 (Ser536, P-p65) (Cell Signaling; #3033). Secondary antibodies were FITC goat anti-mouse Ig and FITC goat anti-rabbit Ig (BD Biosciences). Cell surface markers (BD Biosciences) were Pacific Blue mouse anti-human CD4, Alexa Fluor 700 mouse anti-human CD8, APC mouse anti-human CD19, and PE mouse anti-human CD14. Mouse anti-human HLA-DR was from BD Pharmingen (G46-6). Cyclosporin A (30024), Bay 11-7085 (B5681), and BI-78D3 (B3063) were from Sigma-Aldrich.

Spurlock CF I.I.I., Tossberg J.T., Olsen N.J, & Aune T.M. (2015). Chronic NF-κB activation in CD4+ T cells in rheumatoid arthritis is genetically determined by HLA risk alleles. Journal of immunology (Baltimore, Md. : 1950), 195(3), 791-795.

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5

Cloning and Expression of XFL2 and FcRγ Subunits

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XFL2 cDNA was cloned into pDisplay vector (Invitrogen) as described previously (Guselnikov et al., 2008 (link)). In addition, the complete coding regions of X. laevis FcRγ.a (AF499689) and FcRγ.b cDNAs (EF431895) were cloned into pAP-Tag5 vector (GenHunter). As a result, these constructions encoded XFL2 receptor with N-terminal HA epitope and FcRγ subunits with c-myc epitope at their C-end. 293T cells were transiently transfected with the plasmids. Transfections were carried out using Unifectin 56 (IBCH, Moscow, Russia) according o the manufacturer’s protocol. After 72 h transfection, the cells were used for immunocytochemistry and flow cytometry. The surface expression of XFL2 was analyzed with FACSCanto II cytometer (BD Bioscience): live cells were stained with mouse monoclonal 12CA5 anti-HA antibody (Abcam) and goat anti-mouse Ig-FITC (BD Bioscience). Intracellular expression of FcRγ subunits was detected using microscope Axioscop 2 plus (Carl Zeiss): transfected cells were smeared on glass slides, fixed with acetone and stained with anti-c-myc 9E10 monoclonal antibodies (Abcam) and goat anti-mouse IgG-Tex-asRed (Molecular Probes).

Guselnikov S.V., Grayfer L., De Jesús Andino F., Rogozin I.B., Robert J, & Taranin A.V. (2015). Retention of duplicated ITAM-containing transmembrane signaling subunits in the tetraploid amphibian species Xenopus laevis. Developmental and comparative immunology, 53(1), 158-168.

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Cloning and Expression of XFL2 and FcRγ Subunits

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XFL2 cDNA was cloned into pDisplay vector (Invitrogen) as described previously (Guselnikov et al., 2008 (link)). In addition, the complete coding regions of X. laevis FcRγ.a (AF499689) and FcRγ.b cDNAs (EF431895) were cloned into pAP-Tag5 vector (GenHunter). As a result, these constructions encoded XFL2 receptor with N-terminal HA epitope and FcRγ subunits with c-myc epitope at their C-end. 293T cells were transiently transfected with the plasmids. Transfections were carried out using Unifectin 56 (IBCH, Moscow, Russia) according o the manufacturer’s protocol. After 72 h transfection, the cells were used for immunocytochemistry and flow cytometry. The surface expression of XFL2 was analyzed with FACSCanto II cytometer (BD Bioscience): live cells were stained with mouse monoclonal 12CA5 anti-HA antibody (Abcam) and goat anti-mouse Ig-FITC (BD Bioscience). Intracellular expression of FcRγ subunits was detected using microscope Axioscop 2 plus (Carl Zeiss): transfected cells were smeared on glass slides, fixed with acetone and stained with anti-c-myc 9E10 monoclonal antibodies (Abcam) and goat anti-mouse IgG-Tex-asRed (Molecular Probes).

Guselnikov S.V., Grayfer L., De Jesús Andino F., Rogozin I.B., Robert J, & Taranin A.V. (2015). Retention of duplicated ITAM-containing transmembrane signaling subunits in the tetraploid amphibian species Xenopus laevis. Developmental and comparative immunology, 53(1), 158-168.

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7

Rhesus Macaque MHC Class I Characterization

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MHC gene transferents, a human MHC class I-deficient, Epstein-Barr virus-transformed B lymphoblastoid cell line (721.221) transfected with rhesus macaque MHC class I molecules [32 (link), 33 (link)], and the parental MHC-class I null cell line (721.221) were incubated for 1 hour at 37°C with 20 μl of 1:20 diluted serum. The mouse anti-human MHC class I antibody (clone W6/32; Fisher Scientific) and non-specific mouse IgG1 (BD Biosciences) were included as positive and negative controls, respectively. Cell-bound antibodies were detected with anti-macaque IgG FITC (1B3; NIH Nonhuman Primate Reagent Resource) or goat anti-mouse Ig FITC (polyclonal; BD Biosciences) by incubating antibody-exposed cells for 30 minutes at room temperature. The cells were then fixed with 2% paraformaldehyde and stored at 4°C until being acquired on a LSR II flow cytometer (Becton Dickinson). The data was analyzed using FlowJo software (Tree Star).

Holman N., Weinfurter J.T., Harsla T.R., Wiseman R.W., Belli A.J., Michaels A.J., Reimann K.A., DeMars R.I, & Reynolds M.R. (2017). Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001. PLoS ONE, 12(7), e0179039.

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8

Viral Plaque Assay for Influenza

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The assay was performed as described (Matrosovich et al., 2006 (link)). In brief, MDCK cells (10⁶ per well) were seeded in a 6 well plate to reach 90% confluency the next day. Recombinant virus supernatants were diluted and 100 μl of the selected dilution, to obtain a plaque density of ~20, was added to each well. After incubation for one hour at 37 °C and 5% CO2 cells were washed with PBS once and 2 ml of an overlay containing 2x EMEM (Lonza) and avicel (FMC BioPolymer, Newark, US) in a 1:1 ratio was added. Plates were incubated at 37 °C and 5% CO2. After 40 h, cells were washed with PBS twice and 1 ml of 80% acetone was added. Plates were incubated at −20 °C overnight and virus infection was determined by NP antibody staining. Briefly, NP monoclonal antibody (IgG2a, clone Hb65, American Type Culture Collection, Wesel, Germany) and goat-anti-mouse Ig FITC (BD biosciences, USA) antibody were used to detect NP positive cells. Digital images were taken using ImageQuant TL software (GE Healthcare Life Sciences). The plaque sizes of some viruses were too small to analyze with the software.

Herfst S., Zhang J., Richard M., McBride R., Lexmond P., Bestebroer T.M., Spronken M.I., de Meulder D., van den Brand J.M., Rosu M.E., Martin S.R., Gamblin S.J., Xiong X., Peng W., Bodewes R., van der Vries E., Osterhaus A.D., Paulson J.C., Skehel J.J, & Fouchier R.A. (2020). Hemagglutinin traits determine transmission of avian A/H10N7 influenza virus between mammals. Cell host & microbe, 28(4), 602-613.e7.

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Fitc goat anti mouse ig

Lab products found in correlation

8 protocols using fitc goat anti mouse ig

Immunophenotyping of Neuroblastoma Cells

Immunohistochemical Detection of Adrenergic Receptors and P-gp in Cancer Cells

Immunophenotyping of Neuroblastoma Cells

Flow Cytometric Analysis of PBMCs

Cloning and Expression of XFL2 and FcRγ Subunits

Cloning and Expression of XFL2 and FcRγ Subunits

Rhesus Macaque MHC Class I Characterization

Viral Plaque Assay for Influenza

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