PBMCs, peripheral lymph nodes, spleen and bone marrow cells were labeled with a combination of the following mAbs: CD3 PerCP (SP 34–2), CD4 PerCP (L-200), CD8 PerCP (SK1), CD8 APC (SK1), CD95 FITC (DX2), CD95 APC (DX2), and CD28PE (CD28.2) (BD Pharmingen, San Jose, CA). For chimerism analyses, we used an anti-MHC class I HLA mAb (H38, One Lambda, Inc, CA) reacting specifically with certain MHC class I antigens. The recipient and donor pairs were chosen based on their differential reactivity to this mAb. The fluorescence of the stained samples was analyzed using FACS Calibur and FACS Scan flow cytometers and Cell Quest Software (BD), or FlowJo software. For assessing the effect of CTLA4Ig on memory T cell functions, the monoclonal antibodies anti-CD16, anti-CD95, anti-CD4 and anti-CD8 were used to purify CD4 Tmems (CD16−CD4+CD95+) and CD8 Tmems(CD16−CD8+CD95+) using a FACS Vantage cell sorter (BD Immunocytometry System), then those purified populations were tested for their IFNγ production in ELISPOT assay as previously described (11 (link), 12 (link)). The purity of sorted cells was consistently > 95% as previously described.
Cd95 apc dx2
Manufactured by BD
CD95 APC (DX2) is a fluorochrome-conjugated monoclonal antibody that binds to the CD95 (Fas) antigen. CD95 is a cell surface receptor that plays a role in the regulation of apoptosis. This product is suitable for flow cytometry applications.
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Lab products found in correlation
Cd3 percp sp 34 2, by BD (1 mentions)
Cd28pe cd28.2, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Facsvantage cell sorter, by BD (1 mentions)
Cd3 apch7 sk7, by BD (1 mentions)
Cd3 apc cy7 sk7, by BD (1 mentions)
Cd64 fitc, by BD (1 mentions)
Spss version 21, by IBM (1 mentions)
2 protocols using cd95 apc dx2
1
Multiparametric Flow Cytometry Analysis
2
Multicolor Flow Cytometry Immunophenotyping for Lymphoma
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Coulter (BC; Hialeah, FL) or BD was utilized for the fluorescently labeled antibodies. Antibodies used in the single six-color FC tube for all the specimens (antibody clone designation and supplier) included CD64-FITC (22; BC), CD30-PE (Ber- H83; BD), CD40-PE-Cy5.5 (MAB89; BC), CD20-PE-Cy7 (B9E9; BC), CD95-APC (DX2; BD), and CD3-APC-Cy7 (SK7; BD) or CD3-APC-H7 (SK7; BD). To determine background fluorescence, an appropriate fluorescence minus one control was also utilized. Extensive lymphoma panel was used for FC immunophenotyping which included the following antibodies: CD8-fluorescein isothiocyanate (FITC)/CD4-phycoerythrin (PE)/CD3-Texas Red (ECD)/CD7-PE cyanine 5 (PC5), FITC/CD19-PE/CD10-ECD/CD5- PC5, FITC/CD19-PE/CD10-ECD/CD5-PC5,FMC7-FITC/CD103-PE/CD20-ECD/CD19-PC5,CD38-FITC/CD23-PE/CD19-ECD/CD138-PC5,andCD16+CD56-FITC/CD25-PE/ CD3-ECD/CD14-PC5; all tubes contained CD45-PE cyanine 7 (PC7).The panel was altered, if necessary, based on clinical data, limited material, concurrent morphologic assessment, or previously known lymphoma subtype. Clinical follow-up of greater than six months was required for a negative flow cytometric study without a subsequent biopsy. The data analysis was done using Statistical Package for the Social Sciences (SPSS) version 21. One-way ANOVA was used to compare diagnosis with a number of antibodies used.
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