IgG and IgM alloantibodies were detected by flow cytometry. 1 × 106 F344l splenocytes were suspended in RPMI-1640 medium containing 10% FCS and incubated with diluted recipient LEW (1/50) sera at 37 °C for 30 min. Splenocytes were incubated with the following anti-rat antibodies for 20 min in 0.5% BSA: anti-IgM (MRM-47 Biolegend), anti-IgG (Poly4054 Biolegend), anti-IgG1 (MARG1-2 AbCam), anti-IgG2a (MRG2A-83 Biolegend), anti-IgG2b (MRG2b-85 Biolegend), IgG2c (R2C-23A3 eBioscience), anti-CD3 (14F Biolegend) and anti-CD45 (OX-1 Biolegend). Alloreactivity was analysed by determining the mean fluorescence intensity (MFI) on gated live CD45+ (OX-1 Biolegend) CD3+ (14F Biolegend) cells.
Mrg2b 85
Manufactured by BioLegend
The MRG2b-85 is a lab equipment product from BioLegend. It is designed for use in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mrg2a 83, by BioLegend (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Poly4054, by BioLegend (1 mentions)
Anti cd45, by BioLegend (1 mentions)
Fjk 16, by Thermo Fisher Scientific (1 mentions)
Rm4 5, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd16 cd32 mab 93, by Thermo Fisher Scientific (1 mentions)
Pierce fab preparation kit, by Thermo Fisher Scientific (1 mentions)
Ab8874, by Abcam (1 mentions)
Trustain fcx plus, by BioLegend (1 mentions)
Ab91066, by Abcam (1 mentions)
Poly4064, by BioLegend (1 mentions)
Software, by FlowJo (1 mentions)
3 protocols using mrg2b 85
1
Alloantibody Detection by Flow Cytometry
2
Characterization of Immune Cell Populations Using Fluorescent Antibodies
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Fluorescein isothiocyanate (FITC)-conjugated and/or phycoerythrin (PE)-conjugated anti-mouse CD4 (RM4-5; eBioscience, Inc), anti-mouse CD8α mAb (53-6.7; BD Bioscience), anti-mouse/rat Foxp3 mAb (FJK-16s; eBioscience, Inc), anti-mouse IFN-γ mAb (XMG1.2; eBioscience, Inc), anti-mouse F4/80 mAb (BM8; BioLegend), and anti-rat IgG2b monoclonal antibodies (mAbs) (MRG2b-85; BioLegend) as well as a FITC-conjugated rabbit anti-HSV-1 polyclonal antibody (Dako) were used in flow cytometric analysis and immunohistochemstry. An anti-mouse CD16/CD32 mAb (93; eBioscience, Inc) was used for Fc-blocking in all experiments. For in vivo administration, anti-mouse GITR mAb (DTA-1, rat IgG2b) and anti-mouse CD8α mAb were purified by protein G affinity column chromatography of ascites from BALB/c nude mice intraperitoneally inoculated with a 53-6.7 hybridoma. Purifed rat serum IgG (Sigma) was used as the control antibody for all experiments with DTA-1. The Fab portion of DTA-1 was prepared by using a Pierce Fab Preparation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.
3
Flow Cytometry Characterization of bOECs
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Cells were characterized by flow cytometry 3 weeks after plating (n = 8 WT mice). For analysis, the number of primary bOECs were adjusted to a density of 2x105 cells/ml in phosphate‐buffered saline (PBS)/bovine serum albumin (BSA) solution. TruStain FcX™ PLUS (BioLegend) was added to block non‐specific binding. Characterization was performed using rabbit anti‐p75 nerve growth factor receptor (p75, Abcam, ab8874, RRID:AB_306827) and rat anti‐platelet‐derived growth factor β (PDGFRβ, Abcam, ab91066, RRID:AB_10563302) primary antibodies. P75 positive (with a p75high or a p75low expression) and PDGFRβ negative cells were defined as OECs. PDGFRβ positive and P75 negative cells were identified as stromal cells. Primary antibodies were revealed with the anti‐rabbit phycoerythrin fluorochrome‐conjugated (PE, poly4064, BioLegend, 406408, RRID:AB_10643424) and the anti‐rat Aexafluor 488 fluorochrome‐conjugated antibodies (AF488, MRG2b‐85, BioLegend, RRID:AB_2715913). Data were analyzed using FlowJo software (version 10.3; FlowJo LLC).
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