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]).
Anti igg1 clone rg11 39
Manufactured by BD
The Anti-IgG1 (clone RG11/39.4) is a laboratory reagent used for the detection and analysis of IgG1 antibodies. It is a monoclonal antibody that specifically binds to the IgG1 subclass of immunoglobulin G (IgG). This product can be used in various immunoassay techniques, such as ELISA, to identify and quantify IgG1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Igg2a clone rg7 1.30, by BD (1 mentions)
Igg2b clone rg7 11.1, by BD (1 mentions)
Igm clone g53 238, by BD (1 mentions)
Cd3 clone 1f4, by BioLegend (1 mentions)
Clone g4, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Anti cd45r b220 clone his24, by Thermo Fisher Scientific (1 mentions)
Anti igm clone g53 238, by BD (1 mentions)
3 protocols using anti igg1 clone rg11 39
1
Flow Cytometric Assay for Rat Donor-Specific Antibodies
2
Alloantibody Profiling via Flow Cytometry
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Flow crossmatch was performed using donor (Brown Norway) splenocytes freshly isolated from spleen, macerated through a 50 μm sieve, and washed with R10 after red blood cell lysis with ACK. Cells were suspended, counted, and 500,000 cells were aliquoted into cluster tubes for staining. Lewis rat serum from experimental time points was diluted 1:4 in R10 for a total volume of 50 μL, added to BN donor cells for 30 minutes at room temperature, washed, and stained [14 , 15 ]. Antibodies used include: anti-IgG1 (clone RG11/39.4 BD Bioscience), anti-IgG2a (clone RG7/1.30 BD Pharmingen), anti-IgG2c (clone A92-1 BD Pharmingen), anti-CD3 (1F4 BD Horizon), and anti-CD45R (B220) (clone HIS24 eBioscience). Cells were gated to remove non-singlets, through a lymphocyte gate and then a CD3+ or CD45R+ gate was used in order to perform T lymphocyte or B lymphocyte flow crossmatch, respectively. Mean fluorescence intensity (MFI) was determined for the population of interest.
3
Flow Cytometry Crossmatch for Donor Cells
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Flow crossmatch was performed using donor (Brown Norway) splenocytes freshly isolated from spleen, macerated through a 50 μm sieve, washed with R10 after red blood cell lysis with ACK. Cells were suspended, counted, 500,000 cells were aliquoted into cluster tubes for staining. Lewis rat serum from experimental time points was diluted 1:4 in R10 for a total volume of 50 μL, added to BN donor cells for 30 minutes at room temperature, washed, stained.[14 , 15 (link)] Antibodies used include: anti-IgG1 (clone RG11/39.4 BD Bioscience), anti-IgG2a (clone RG7/1.30 BD Pharmingen), anti-IgG2b (clone RG7/11.1 BD Pharmingen), anti-IgG2c (clone A92-1 BD Pharmingen), anti-IgM (clone G53-238 BD Pharmingen), anti-CD3 (1F4 BD Horizon), anti-CD45R (B220) (clone HIS24 eBioscience). Cells were gated to remove non-singlets through a lymphocyte gate then a CD3+ or CD45R+ gate was used in order to perform T lymphocyte or B lymphocyte flow crossmatch, respectively. Mean fluorescence intensity (MFI) was determined for the population of interest.
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