Chemotaxis assay.

<p>Chemokine-induced migration of infected mature MDDC was tested 48 h and 72 h after infection in a chemotaxis assay (n = 6). Entire populations that migrated toward CCL9 and CCL21 were collected after incubation and counted during one minute by flow cytometry. The number of migrated DC is depicted in percent of the initial input. Subsequently, infected (MVA and MVA-B) and uninfected MDDC that migrated in the chemotaxis assay were collected and co-cultured with CFSE labeled autologous lymphocytes at a 1∶40 ratio (5×10³ MDDC: 2×10⁵ lymphocytes/well). After a period of 6 days, proliferation was tested by flow cytometry. (A) Percentage of CCL19 specific migration of MVA-B-infected MDDC 48 hours after infection. Error bars represent the mean (±SEM) from 6 independent experiments with 6 different HIV-1 patients (n = 6) performed in triplicates. (B) Percentage of CCL19 specific migration of MVA and MVA-B-infected and uninfected MDDC (Mock) 48 and 72 h post-infection and maturation. Error bars represent the mean (±SEM) from 3 independent experiments with 3 different HIV-1 patients (n = 3) performed in triplicates. (C) Representative flow cytometric plots showing CFSE dilution in gated CD3⁺ CD8⁺ lymphocytes after co-culture with MVA and MVA-B infected MDDC that were able to migrate in the chemotaxis assay. One representative result of 2 independent experiments is shown. Error bars represent the mean (±SEM) (n = 3). (D) Percentage of CFSE^low cells in the CD3⁺ CD8⁺ gate, observed in one sample. Error bars represent the SEM from the mean triplicates (***) p = 0.005.