Serum was isolated from peripheral blood draws at specific intervals from nonhuman primates (n=6) prior to and after skin transplantation for donor specific antibody measurement. Donor specific antibody was measured by flow crossmatch as previously described (Kim et al., 2014 (link)). Donor peripheral blood mononuclear cells (PBMCs) were blocked with 1:1000 dilution of goat IgG whole molecule solution (005-000-003, Jackson ImmunoResearch Laboratory, Inc. West Grove, PA), washed with 2% fetal bovine serum (FBS) in phosphate buffered saline (PBS), incubated with recipient serum at a 1:50 dilution, and washed again. Cells were then stained with fixable blue dye (L23105, Invitrogen, Waltham, MA), FITC-anti-monkey IgG (polyclonal, 5230-0422, Seracare, Milford, MA), PE-anti-human CD20 (clone 2H7, BD Biosciences, San Jose, CA), and PerCP-Cy5.5-anti-human CD3 (clone SP34-2, BD Biosciences). Cells underwent one more wash and were fixed with stabilizing fixative (338036, BD Biosciences). The mean fluorescence intensity (MFI) of recipient IgG on donor CD3+ T cells and CD20+ B cells were measured using a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo version 10 software (BD Biosciences).
Pe anti human cd20 clone 2h7
Manufactured by BD
PE-anti-human CD20 (clone 2H7) is a fluorescently-labeled antibody that specifically binds to the CD20 antigen expressed on human B cells. This product can be used for the identification and enumeration of B cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (1 mentions)
L23105, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Merck Group (1 mentions)
Guava flow cytometer, by Merck Group (1 mentions)
Inspire software, by Merck Group (1 mentions)
Guavasoft software, by Merck Group (1 mentions)
Amnis imagestream, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
2 protocols using pe anti human cd20 clone 2h7
1
Detecting Donor-Specific Antibodies in Transplant
2
Fluorescent Labeling of ABX and Rituximab
Check if the same lab product or an alternative is used in the 5 most similar protocols
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ABX and rituximab were labeled with Alexa-fluor 488 (Thermo Scientific, Rockford, IL) by incubation with 1 mg of protein for 60 minutes at room temperature and unbound label was removed by size exclusion column chromatography. Daudi cells were incubated for 30 minutes with labeled AR160, PE anti-human CD19 (Clone HIB19), and PE anti-human CD20 (Clone 2H7) (BD Pharmingen, Franklin Lakes, NJ) at 4 °C. The cells were washed 2x in FACS buffer (1x PBS and 0.5% BSA with 0.1% NaAzide). The cells were run and data was collected on a Guava flow cytometer (Millipore, Billerica, MA). Five thousand events were collected and flow cytometery data was analyzed using GuavaSoft software (Millipore, Billerica, MA) and percentages of AR160+, CD19+ and CD20+ cells were determined. Labeled ABX and PE anti-human CD19 (Clone HIB19)/Alexa-fluor 488 AR160 labeled Daudi cells were collected by Amnis ImageStream (Millipore, Billerica, MA). Inspire software (Millipore, Billerica, MA) was used to analyze data and collect pictures.
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