DSA in humans were analyzed as described previously, using the single antigen bead Luminex (One Lambda) assay (23 (link),24 (link)). Rat DSA measurement was performed as described elsewhere (25 (link),26 (link)). Briefly, donor (BN, RT1n) splenocytes were freshly isolated from spleen macerated through a 50-μm sieve and washed after red blood cell lysis. Cells were resuspended, counted and aliquoted into cluster tubes for staining. Fifty microliters of 1:4 dilution of serum or plasma from 1 week posttransplant (or similar time points for control or transfusion only rats) were incubated for 30 min at 37°C, then washed and stained. Flow cytometry was performed on a BD LSR II at the Immunology Core of the WNPRC, UW-Madison and data analyzed using FlowJo (TreeStar, Inc., Ashland, OR). Cells were gated to remove nonsinglets, through a lymphocyte gate, then a CD3-positive gate to remove cells that had an FcR, which could skew analyses. Mean fluorescence intensity was determined for the populations of interest. Antibodies used were anti-IgG1 (clone RG11/39.4; BD Biosciences, San Diego, CA), IgG2a (clone RG7/1.30; BD Biosciences), IgG2b (clone RG7/11.1; BD Biosciences), IgG2c (biotinylated, clone A92-1, BD Biosciences), IgM (clone G53-238; BD Biosciences) and CD3 (clone 1F4 [BioLegend, San Diego, CA] or clone G4.18 [BD Biosciences]).
Clone g4
Manufactured by BD
Sourced in United States
The Clone G4.18 is a laboratory instrument designed for general use in research settings. It functions as a versatile tool for various scientific applications. The core purpose of this product is to assist researchers in their work, though its specific intended uses are not detailed here.
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Lab products found in correlation
Clone ox 35, by BD (4 mentions)
Clone fjk 16s, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (3 mentions)
Igg2a clone rg7 1.30, by BD (2 mentions)
Igg2b clone rg7 11.1, by BD (2 mentions)
Igm clone g53 238, by BD (2 mentions)
Cd3 clone 1f4, by BioLegend (2 mentions)
Lsr 2, by BD (2 mentions)
Clone 145 2c11, by BD (2 mentions)
Cyquant direct cell proliferation, by Thermo Fisher Scientific (2 mentions)
Spectramax m2e spectrofluorometer, by Molecular Devices (2 mentions)
Softmax pro software v5, by Molecular Devices (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fix perm concentrate, by BD (2 mentions)
Clone ebio17b7, by Thermo Fisher Scientific (2 mentions)
Anti igg1 clone rg11 39, by BD (1 mentions)
Deoxyribonuclease, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Fujifilm (1 mentions)
Facsmelody, by BD (1 mentions)
8 protocols using clone g4
1
Flow Cytometric Assay for Rat Donor-Specific Antibodies
2
Isolation and Characterization of Immune Cells
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The tissue of transplanted area was minced with a scissors, after which the tissue fragments were incubated for 1 h at 37 ºC in Dulbecco's Modified Eagle Medium (DMEM: Invitrogen) in the presence of 0.2% collagenase (Wako Chemicals, Cell dissociation grade), and 25 μg/ml deoxyribonuclease (Sigma-Aldrich). The suspension was filtered through a cell strainer (Falcon 2350, 70 μm) to remove debris and tissue fragments, after which the cells were pelleted by centrifugation at 300 g for 5 min at room temperature. Next, the cells were resuspended in calcium- and magnesium-free Hank's Balanced Salt Solution (HBSS) (Fujifilm) supplemented with 2% FBS, 10 mM HEPES and 1% penicillin/streptomycin (P/S). The cells were stained with immune cell markers as described below; PE conjugated mouse anti-rat CD3 (BD Pharmingen, Clone G4.18, Catalog No:554833), FITC conjugated mouse anti-rat CD4 (BD Pharmingen, Clone OX-35, Catalog No:554837), Catalog No:561588), PE conjugated mouse anti-rat CD11b/c (BD Pharmingen, Clone OX-42, Catalog No:554862), and Alexa Fluor® 647 conjugated mouse anti-rat CD163 (Bio-Rad Laboratories, Inc., Clone ED2, Catalog No: MCA342A647). Flow-cytometric analysis was performed on FACSMelody (BD Biosciences), and the data were analyzed using FlowJo software (BD Biosciences).
3
Quantifying T-Cell Proliferation Assay
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To evaluate T-cell proliferation, 96-well microtiter plates were coated overnight with anti-mouse CD3 antibody (100 μl/well of 1 μg/ml solution of Clone 145–2C11; BD Biosciences) or anti-rat CD3 antibody (100 μl/well of 1 μg/ml solution of Clone G4.18; BD Biosciences) and then washed. Spleen cell suspensions from Cohort 4 animals were separated from red blood cells (RBC) using Ficoll-Paque Plus (GE Healthcare) density gradients. The isolated cells were then re-suspended in complete RPMI 1640 medium (RPMI containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin; Gibco ThermoFisher Scientific) at 5 × 106 cells/ml. From this suspension, 100 μl of cells (5 × 105/well) were added to appropriate wells, and the plates incubated at 37 °C (in 5% CO2) for up to 72 h. Changes in total cell number, indicative of proliferation, were measured using a fluorescent nucleic acid stain assay (CyQuant Direct Cell Proliferation; ThermoFisher Scientific, Grand Island, NY) according to manufacturer instruct-tions. The fluorescent signal (485 nm excitation/535 nm emission), directly proportional to live cell number and thereby an index of proliferation, was measured using a Spectramax M2e spectrofluorometer and associated Softmax Pro software v5.0 (Molecular Devices).
4
Quantifying T-Cell Proliferation Assay
5
Phenotypic Analysis of Splenic T-Cells
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For the performance of the phenotypic analyses, the cultured splenic T-cells were incubated with specific antibodies targeting T-cell surface markers. This included fluorescein (FITC)-conjugated anti-CD3 (1:200, clone G4.18, #554832, BD Bioscience, Franklin Lakes, NJ, USA) and phycoerythrin (PE)-conjugated anti-CD4 (1:200, clone OX-35, #554838, BD Bioscience). For the identification of Treg and Th17 cells, the cells were stained with peridinin-chlorophyll-protein (PerCP)–Cyanine5.5 conjugated anti-FoxP3 (1:100, clone FJK-16s, #45-5773-82, Thermo Fisher Scientific) for Treg cells or allophycocyanine (APC)-conjugated anti-retinoic-acid-receptor-related orphan nuclear receptor gamma (RORγt) (1:1000, clone 4G419, NBP2-24449, Novus Biologicals, Centennial, CO, USA) for Th17 cells, employing the FoxP3/Transcription Factor Staining Buffer Set (00-5523-00, eBioscience) [17 (link)]. The data were acquired using a 4-color flow cytometer (FACS Calibur; BD Bioscience, Franklin Lakes, NJ, USA) and were subsequently analyzed using FlowJo v10 software (BD Biosciences).
6
Immunophenotyping of Th17 and Treg Cells
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Single-cell suspensions of spleen were generated as has been described previously (Taylor et al., 2018 (link)). Blood was obtained from SS and SR rats, which had taken 20% fructose or tap water for 4 weeks. PBMCs were separated by Ficoll-Paque PLUS gradient centrifugation (GE Healthcare, Chicago, IL, USA). Briefly, phenotypic and intracellular analyses were performed by incubating cells with antibodies for T cell surface markers – fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat CD3 (1:100; clone G4.18, #554832; BD Biosciences, Franklin Lakes, NJ, USA) and PE-conjugated mouse anti-rat CD4 (1:100; clone OX-35, #554838; BD Biosciences) – for 30 min on ice in the dark. After washing, the cells were fixed and permeabilized using fix/perm concentrate (BD Biosciences) before incubation with antibodies for intracellular staining of APC rat anti-rat IL-17A (1:100; clone eBio17B7, #17-7177-81; Thermo Fisher Scientific) to identify Th17 cells and PerCP-cyanine5.5 rat anti-rat FOXP3 (1:100; clone FJK-16s, #14-5773-82; Thermo Fisher Scientific) to identify Treg cells. Cells were then washed and run through a four-color flow cytometer (FACSCalibur; BD Biosciences) and data were collected using CellQuest or FlowJo v10.0 software. An example of the flow cytometry gating strategy used is shown in Fig. S3 .
7
Measurement of DSA Antibody Isotypes
8
Immunophenotyping of T Cell Subsets
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The single-cell suspension method of PBMCs was described in our previous study [13 (link)]. The single-cell suspension method of the spleen and kidney was described in previous studies [23 (link), 24 ]. Rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) for sacrifice, and then, the serum, spleen, and kidney were taken and stored at -80°C in liquid nitrogen until further studies. Monoclonal antibodies FITC mouse anti-rat CD3 (1 : 100, clone G4.18, #554832, BD Bioscience, Franklin Lakes, NJ, USA) and PE mouse anti-rat CD4 (1 : 100, clone OX-35, #554838, BD Bioscience) as T cell surface markers stained the PBMCs, spleen, and kidney from WKY and SHR. CD3 and CD4 were used to identify T helper (Th) cells. To identify Th17 cells or Treg cells, the stained CD3 and CD4 cells were washed and fixed and then were permeabilized using fix/perm concentrate (BD Bioscience). The cells were incubated with the monoclonal antibody APC rat anti-rat IL-17A (1 : 100, clone eBio17B7, #17-7177-81, Thermo Fisher Scientific) for Th17 cells or PerCP-Cyanine5.5 rat anti-rat FoxP3 (1 : 100, clone FJK-16s, #14-5773-82, Thermo Fisher Scientific) for Treg cells. After incubation, the cells were washed and run through a four-color flow cytometer (FACSCalibur; BD Biosciences). Data were collected using CellQuest.
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