All the fluorochrome-conjugated mAbs used in this study were purchased from Biolegend or eBioscience unless otherwise stated. Supernatants from the hybridomas (2C11, GK1.5, TIB105, 10.2.16, 2.4G2) producing mAbs (to CD3, CD4, CD8, MHC-class II I-Ag7 and Fc receptor, respectively), used for cell purification or stimulation, were generously provided by the late Charles Janeway Jr. (Yale University). Magnetic beads conjugated with goat anti-mouse IgG, goat anti-mouse IgM or goat anti-rat IgG were purchased from QIAGEN. RPMI-1640 medium and heat-inactivated FCS were purchased from Invitrogen and Gemini, respectively.
Fluorochrome conjugated mabs
Manufactured by BioLegend
Fluorochrome-conjugated monoclonal antibodies (mAbs) are laboratory reagents used for the detection and quantification of specific proteins or cellular markers in various biological samples. These antibodies are conjugated with fluorescent dyes, enabling the visualization and analysis of target cells or molecules using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg, by Qiagen (1 mentions)
Goat anti mouse igm, by Qiagen (1 mentions)
Goat anti rat igg, by Qiagen (1 mentions)
Heat inactivated fcs, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by FlowJo (1 mentions)
Lsrfortessa, by BD (1 mentions)
3 protocols using fluorochrome conjugated mabs
1
Detailed Immunological Reagents Protocol
2
Quantification of Murine Immune Cell Populations
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Blood from the submandibular vein was collected into 200 μl of sodium citrate (130 mM). Cells were washed in 3 ml HBSS and stained for 1 h at 4°C in the dark with designated cocktails of fluorochrome-conjugated antibodies. After staining, red blood cells were lysed by incubation for 10 min on ice with 3 ml of ammonium chloride lysis buffer (150 mM NH4Cl, 10 mM NaHCO3, and 1.2 mM EDTA pH 7.2). Samples were washed three times with HBSS and analyzed by use of a Becton-Dickson LSRII flow cytometer (San Jose, CA, United States) followed by analysis with FlowJo software (Ashland, OR, United States). In designated experiments, reference beads (AccuCount PE- or APC-conjugated EasyComp fluorescent particles 3.0–3.4 μm, Spherotech, Lake Forest, IL, United States) were added to samples immediately before cytometric analysis to assess absolute cell numbers. Fluorochrome-conjugated mAbs were obtained from BioLegend and were specific for CD3 (17A2 or 145-2C11), CD4 (GK1.5), CD25 (PC61, 7D4, and 3C7), CD45.1 (A20), CD45.2 (104), CD69 (H1.2F3), Histag (J095G46), Neuropilin-1 (NRP-1, 3E12), TCR Vβ11 (KT11), TCR Vα3.2 (RR3-16), TCR Vβ5.1,5.2 (MR9-4), and TCR Vα2 (B20.1).
3
Flow Cytometry Cell Staining
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We resuspended cells in staining buffer (PBS with 10% FBS) with 1 × 106 cells/ml, and blocked the nonspecific staining by anti-CD16/32 for 10 min on ice. We then stained the cells with monoclonal antibodies (mAbs) at 4°C for 30 min, and washed them with PBS. We measured marker expression on the BD LSRFortessa (BD) and analyzed with FlowJo software. We purchased fluorochrome-conjugated mAbs and the corresponding isotype controls from Biolegend, BD Biosciences, or eBioscience, as indicated in Supplementary Methods file .
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