![Figure 1. Effect of FcμR on Fas-mediated apoptosis in Jurkat cells. (A) Jurkat cells transduced with the bicistronic retroviral construct containing both human FcμR and GFP cDNAs (FcμR/GFP) or only GFP cDNA (GFP) as a control were incubated with biotin-labeled, anti-FcμR/Toso mAbs (HM7 [γ2bκ], HM14 [γ1κ] or 1E4 [γ1κ; Abnoca Corp]) or isotype-matched control mAbs (γ2bκ or γ1κ) as described.2,4,5 The bound mAbs were detected by addition of phycoerythrin-labeled streptavidin (PE-SA). For IgM binding, cells were incubated with myeloma or hybridoma-derived IgM of both human and mouse origin at 10 μg/mL, washed, and then incubated with biotin-labeled, rat anti–mouse μ mAb (clone 332.12) for mouse IgM or mouse anti–human μ mAb (clone SA-DA4.4) for human IgM. Biotin-labeled, control mAbs of the same isotype (rat γ2bκ or mouse γ1κ) were also included. Stained cells were analyzed by Accuri C6 Flow Cytometer. Because all staining profiles with control mAbs were indistinguishable, only the result with biotin-labeled mouse IgG1 control mAb is shown for simplicity. Note the expression of FcμR/Toso on FcμR/GFP transductants as determined by reactivity with 3 different mAbs and by IgM-ligand binding. (Only the binding result with mouse IgMλ hybridoma [B1–8; anti-nitrophenyl antibody] is shown.) (B) Both cell lines were cultured at 37°C for 12 (top panel) and 20 hours (bottom panel) without or with agonistic mouse anti–human Fas mAb of IgMκ (CH11; 10 ng/mL; Millipore) or IgG3κ isotype (2R2; 1μg/mL; Invitrogen) or with a recombinant human FasL (superFasL; Enzo Life Sciences) at 3 different protein concentrations (1, 10 and 100 ng/mL). Cells were stained with 7-aminoactinomycin D (7-AAD) and allophycocyanin (APC)–labeled annexin V to identify early (annexin V+/7-AAD−) and late (annexin V+/7-AAD+) apoptotic and dead (annexin V−/7-AAD+) cells by flow cytometry.2 Note the resistance of the FcμR/GFP transductant to Fas-mediated apoptosis by IgM mAb, but not by IgG3 mAb and FasL. These experiments were performed 3 times with essentially the same results.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/119/7/10.1182_blood-2011-09-380782/4/zh89991184760001.jpeg?Expires=1735818649&Signature=WPZRU4U4ERKiGwSoItuQmFXmgPRnS51uwR4Wri0LWWoscY2NszjSRiErnMexs4tGvxJVGW4C3fTJbMQVShCwCFeYbLXkjizSdVDSY18GhQF-c3udpAFcj71KMYgIi3mi24wz~BaIHUWSt4dXaZL~NrC6ITYN0p0h3Lu7Oz3Fq2q9peOwELcEcVWJgyWPh1PtTNEIAlYvsQLEyr9oFRYu4leBFhvOcB~xkPkXakEBFl9OrgaDDs0Z3WBAA70fCC7MOwv~RMmSH-fPEExblvA-FJi1PNcneTokHn2mwIeRYCAKLZJUwfJ9agVw9IjBuIO7C8KxbYI3nYzH6DXV-FS3FQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of FcμR on Fas-mediated apoptosis in Jurkat cells. (A) Jurkat cells transduced with the bicistronic retroviral construct containing both human FcμR and GFP cDNAs (FcμR/GFP) or only GFP cDNA (GFP) as a control were incubated with biotin-labeled, anti-FcμR/Toso mAbs (HM7 [γ2bκ], HM14 [γ1κ] or 1E4 [γ1κ; Abnoca Corp]) or isotype-matched control mAbs (γ2bκ or γ1κ) as described.2,4,5 The bound mAbs were detected by addition of phycoerythrin-labeled streptavidin (PE-SA). For IgM binding, cells were incubated with myeloma or hybridoma-derived IgM of both human and mouse origin at 10 μg/mL, washed, and then incubated with biotin-labeled, rat anti–mouse μ mAb (clone 332.12) for mouse IgM or mouse anti–human μ mAb (clone SA-DA4.4) for human IgM. Biotin-labeled, control mAbs of the same isotype (rat γ2bκ or mouse γ1κ) were also included. Stained cells were analyzed by Accuri C6 Flow Cytometer. Because all staining profiles with control mAbs were indistinguishable, only the result with biotin-labeled mouse IgG1 control mAb is shown for simplicity. Note the expression of FcμR/Toso on FcμR/GFP transductants as determined by reactivity with 3 different mAbs and by IgM-ligand binding. (Only the binding result with mouse IgMλ hybridoma [B1–8; anti-nitrophenyl antibody] is shown.) (B) Both cell lines were cultured at 37°C for 12 (top panel) and 20 hours (bottom panel) without or with agonistic mouse anti–human Fas mAb of IgMκ (CH11; 10 ng/mL; Millipore) or IgG3κ isotype (2R2; 1μg/mL; Invitrogen) or with a recombinant human FasL (superFasL; Enzo Life Sciences) at 3 different protein concentrations (1, 10 and 100 ng/mL). Cells were stained with 7-aminoactinomycin D (7-AAD) and allophycocyanin (APC)–labeled annexin V to identify early (annexin V+/7-AAD−) and late (annexin V+/7-AAD+) apoptotic and dead (annexin V−/7-AAD+) cells by flow cytometry.2 Note the resistance of the FcμR/GFP transductant to Fas-mediated apoptosis by IgM mAb, but not by IgG3 mAb and FasL. These experiments were performed 3 times with essentially the same results.
