Fig. 2.
Fig. 2. Transduced and mock-transduced CD34+ progenitors differentiate into dendritic cells expressing typical morphology and dendritic cell markers CD83 and p55. CD34+ cord blood progenitors were cultured in complete IMDM-20% FCS with c-kit-ligand, GM-CSF, and TNFα and transduced on day 3 of culture. Cells were recultured after transduction in original media with replenished cytokines for an additional 8 to 10 days. Washed cells were cytofluorographically sorted into two populations based on the presence or absence of surface mCD2 and were then processed for immunostaining. To enrich for dendritic cells only, plastic nonadherent cells showing high forward and side scatter properties were collected. Expression of murine CD2, p55, and CD83 was detected by immunocytochemistry on cytocentrifuged, acetone-fixed cells using a standard alkaline phosphatase-based staining method. The mock-transduced population served as its own control for murine CD2 staining in the transduced population. An IgG isotype control for CD83 and p55 staining is illustrated for each of the mock-transduced and transduced populations. CD40 and S100 staining were also comparable in each population (not shown).

Transduced and mock-transduced CD34+ progenitors differentiate into dendritic cells expressing typical morphology and dendritic cell markers CD83 and p55. CD34+ cord blood progenitors were cultured in complete IMDM-20% FCS with c-kit-ligand, GM-CSF, and TNFα and transduced on day 3 of culture. Cells were recultured after transduction in original media with replenished cytokines for an additional 8 to 10 days. Washed cells were cytofluorographically sorted into two populations based on the presence or absence of surface mCD2 and were then processed for immunostaining. To enrich for dendritic cells only, plastic nonadherent cells showing high forward and side scatter properties were collected. Expression of murine CD2, p55, and CD83 was detected by immunocytochemistry on cytocentrifuged, acetone-fixed cells using a standard alkaline phosphatase-based staining method. The mock-transduced population served as its own control for murine CD2 staining in the transduced population. An IgG isotype control for CD83 and p55 staining is illustrated for each of the mock-transduced and transduced populations. CD40 and S100 staining were also comparable in each population (not shown).

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