To examine the immunogenicity associated with hepatic gene transfer of AAV2 vectors, we enumerated the T cell, B cell, and T-reg cells in hemophilia B mice that received gene therapy (n = 5 per group). Briefly, peripheral blood from hemophilia B mice was collected 9 weeks after gene transfer. After RBC lysis (155 mM NH4Cl, 12 mM NaHCO3, and 0.1 mM EDTA), samples were incubated with a combination of FITC-labeled anti-CD3, PE-labeled anti-CD8, PerCP-labeled anti-CD4, and APC-labeled anti-CD19 (BD Biosciences) antibodies for 30 min at room temperature, and percentage CD3+, CD4+, CD8+, and CD19+ cells were assessed by flow cytometry (BD Accuri C6 Plus). To profile the T-reg cells in murine splenocytes, ~2 million cells were stained with PerCP-labeled anti-CD4 and APC-labeled anti-CD25 and PE-conjugated Foxp3 antibodies as per the manufacturer’s protocol (BD Biosciences).
Apc labeled anti cd19
Manufactured by BD
APC-labeled anti-CD19 is a flow cytometry reagent that binds to the CD19 surface antigen expressed on B cells. It is used for the identification and enumeration of B cells in cell samples.
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3 protocols using apc labeled anti cd19
1
Immunogenicity of Hepatic AAV2 Gene Therapy
2
Assessing Immunogenic Response to AAV2 Gene Therapy
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To examine the immunogenicity associated with hepatic gene transfer of AAV2 vectors, we enumerated the T cell, B cell, and T-reg cells in hemophilia B mice that received gene therapy (n = 5 per group). Briefly, peripheral blood from hemophilia B mice was collected 9 weeks after gene transfer. After RBC lysis (155 mM NH4Cl, 12 mM NaHCO3, and 0.1 mM EDTA), samples were incubated with a combination of FITC-labeled anti-CD3, PE-labeled anti-CD8, PerCP-labeled anti-CD4, and APC-labeled anti-CD19 (BD Biosciences) antibodies for 30 min at room temperature, and percentage CD3+, CD4+, CD8+, and CD19+ cells were assessed by flow cytometry (BD Accuri C6 Plus). To profile the T-reg cells in murine splenocytes, ∼2 million cells were stained with PerCP-labeled anti-CD4 and APC-labeled anti-CD25 and PE-conjugated Foxp3 antibodies as per the manufacturer's protocol (BD Biosciences).
3
Phenotypic Analysis of Leukemia Cells
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Raji and Ramos cells were treated with 6 μM TG101209 for 96 h and then incubated with FITC-labeled anti-CD38 antibody (#555459), PE-labeled anti-CD138 (#552026), APC-labeled anti-CD19 (#555415) and PE-labeled anti-CD10 (#555375) (BD Pharmingen, CA), and the percentage of positive cells was examined by flow cytometry. For morphological analysis, cytospin slides of each sample were stained with Wright-Giemsa staining and observed under a Nikon inverted light microscope (Eclipse TE300; Nikon Corporation, Tokyo, Japan) at 1000 × magnification. Physical parameters (forward scatter [FS] and side scatter [SS]) in Raji and Ramos cell lines were also analyzed by flow cytometry.
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