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.
Poly4054
Manufactured by BioLegend
Sourced in United States
Poly4054 is a high-quality polymer reagent designed for use in various biomedical and biotechnology applications. It serves as a versatile tool for researchers and scientists working in fields such as immunology, cell biology, and molecular biology. The core function of Poly4054 is to facilitate efficient and reliable labeling, detection, and capture of target molecules in a wide range of experimental assays and protocols.
Automatically generated - may contain errors
Lab products found in correlation
Mrg2a 83, by BioLegend (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Mrg2b 85, by BioLegend (1 mentions)
Anti cd45, by BioLegend (1 mentions)
Hp6017, by BioLegend (1 mentions)
Apc conjugated mouse anti human igg fc antibody hp6017, by BioLegend (1 mentions)
Ab150353, by Abcam (1 mentions)
Anti tubulin, by Cell Signaling Technology (1 mentions)
Silverquest kit, by Thermo Fisher Scientific (1 mentions)
Anti ptbp1, by Cell Signaling Technology (1 mentions)
Rb6 8c5, by BioLegend (1 mentions)
4 protocols using poly4054
1
Alloantibody Detection by Flow Cytometry
2
Evaluating PVRIG-PVRL2 Antagonism Assays
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For rat anti-mouse PVRIG monoclonal antibody (Clone 1) binding assay, 2 × 105 293T-mouse PVRIG cells were incubated with different concentrations of antibodies (Clone 1), and then the binding frequency was detected using APC-conjugated goat anti-rat IgG antibody (Poly4054, Biolegend, San Diego, USA). For assessing PVRIG-PVRL2 antagonistic activity, 2 × 105 293T-mouse PVRIG cells were incubated with different concentrations of antibodies (Clone 1) and 10 μg/mL mouse PVRL2-hFc fusion protein. APC-conjugated mouse anti-human IgG Fc antibody (HP6017, Biolegend, San Diego, USA) was used to detect the binding frequency of mCD112-hFc fusion protein.
For mouse anti-human PVRIG monoclonal antibody (Clone 2) binding assay, 2 × 105 293T-human PVRIG cells were incubated with different concentrations of antibodies (Clone 2), and then the binding frequency was detected using APC-conjugated goat anti-mouse IgG antibody (Poly4053, Biolegend, San Diego, USA). For assessing PVRIG-PVRL2 antagonistic activity, 2 × 105 293T-human PVRL2 cells were incubated with different concentrations of antibodies (Clone 2) and 10 μg/mL human PVRIG-Fc fusion protein. APC-conjugated mouse anti-human IgG Fc antibody (HP6017, Biolegend, San Diego, USA) was used to detect the binding frequency of human PVRIG-Fc fusion protein.
For mouse anti-human PVRIG monoclonal antibody (Clone 2) binding assay, 2 × 105 293T-human PVRIG cells were incubated with different concentrations of antibodies (Clone 2), and then the binding frequency was detected using APC-conjugated goat anti-mouse IgG antibody (Poly4053, Biolegend, San Diego, USA). For assessing PVRIG-PVRL2 antagonistic activity, 2 × 105 293T-human PVRL2 cells were incubated with different concentrations of antibodies (Clone 2) and 10 μg/mL human PVRIG-Fc fusion protein. APC-conjugated mouse anti-human IgG Fc antibody (HP6017, Biolegend, San Diego, USA) was used to detect the binding frequency of human PVRIG-Fc fusion protein.
3
Protein Visualization and Antibody Western Blot Protocols
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All protein visualization procedures were performed according to standard protocols. For silver staining we used the SilverQuest kit (LC6070, Invitrogen). The following in-house generated monoclonal antibody supernatants were used in Western blots at a 1:10 dilution: anti-Roquin-1/2, cl. 3F12; anti-Regnase-1, cl. 15D11; anti-pan-Ago, cl. MAGO3-5, anti-Nufip2, cl. 23G8, anti-pan-YTHDF, cl. 17F2; anti-GFP, cl. 3E5-111. Commercial antibodies used for Western blots were: anti-Fxr1, polyclonal, 4173, 1:1000, (Cell Signaling); anti-Fxr2, cl. D85D6, 7098, 1:1000 (Cell Signaling); anti-TTP, cl. TP6, 1:1000 (Sigma); anti-Gapdh, cl. 6C5, CB1001, 1:10,000 (Calbiochem), anti-Celf1, cl. 850717, MAB9388, 0.5 µg/ml (R&D SYSTEMS); anti-RBMS1, cl. EPR9825(B), ab150353, 1:5000 (abcam); anti-CPEB4, polyclonal, 25342-1-AP, 1:750 (Proteintech); anti-Ptbp1, 1:1000, 8776 (Cell Signaling); anti-tubulin, 1:1000, 86298 (Cell Signaling); goat anti-rat antibody, cl. Poly4054, 1:200 (Biolegend); goat anti-mouse antibody, polyclonal, 554001, 1:400 (BD Bioscience) and anti-Ptbp1, (1:1000, 8776, Cell Signaling). Proteins were visualized by staining with anti-rat (1:3000, 7077, Cell Signaling) or anti-mouse (1:3000, 7076, Cell Signaling) secondary antibodies conjugated to HRP.
4
Gr1+ Myeloid Cell IHC in Colon Tumor Mice
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The colon or colorectal tumor tissues from mouse models were collected and fixed in 10% formalin overnight and embedded in paraffin. Formalin-fixed paraffin-embedded tissue was cut into 4 μm sections. The sections were stained using standard protocols for xylene and an alcohol gradient for deparaffinization. After antigen retrieval and unmarking procedures, the primary rat anti-mouse Gr1 antibody (1 ng/ml in 50 µl volume; RB6-8C5; Biolegend, San Diego, CA, USA) was incubated and stained. Sections were incubated with HRP goat anti-rat IgG antibody (2.0 µg/ml; Poly4054; Biolegend, San Diego, CA, USA) for 40 min and then developed with an Ultravision DAB Plus Substrate Detection System (TA-125-QHDX, Thermo Fischer Scientific, Waltham, MA, USA) for 2–5 min at room temperature, followed by hematoxylin staining, dehydration and coverslipping with Permount. Immunohistochemistry (IHC) slides were scanned with a Pannoramic Digital Slide Scanner (SDHISTECH, Budapest, Hungary), and images were cropped from virtual slides in Pannoramic Viewer.
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