Figure 1. BDCA3 DCs have a more mature... | Rockefeller University Press

Figure 1.

$Figure 1. BDCA3+ DCs have a more mature phenotype than BDCA1+ DCs. (A–C) Shown are representative plots for BDCA1+ DCs (A), BDCA3+ DCs (B), and pDCs (C) after isolation (representative of n > 20 donors). (D) Percentage of contaminating cells in isolated DC subsets. Isolated DC fractions were labeled for contaminating cell subsets with antibodies against CD14, CD123, BDCA1, or BDCA3. Live cells were gated on the above markers and the percentage of contaminating subsets was determined. Data are the mean ± SD (n = 7 independent experiments). NA, not applicable (below limit of detection). (E) Phenotype of DC subsets in freshly isolated PBMCs. DCs in PBMCs were labeled for the indicated markers and analyzed by flow cytometry. Isotype background fluorescence was subtracted from the mean fluorescence intensity (MFI), and MFI was normalized to unstimulated BDCA1+ DCs. Data are the mean ± SD (n = 6 independent experiments). (F) Phenotype of isolated DC subsets after overnight culture in the presence or absence of TLR7/8 L. After overnight culture, DCs were labeled for the indicated markers and analyzed by flow cytometry. Isotype background fluorescence was subtracted from the MFI, and MFI was normalized to unstimulated BDCA1+ DCs. Data are the mean ± SD (n = 6 independent experiments). (G) Endocytic capacity of isolated DC subsets. Overnight cultured isolated DCs were pulsed with 5 µg/ml Alexa Fluor 488 ovalbumin for 20 min at 37 or 4°C, and ovalbumin uptake was analyzed by flow cytometry. Shown is one representative experiment of three. (H) Cytokine production by isolated DC subsets after overnight culture in the presence or absence of TLR7/8 L. Supernatants were collected and analyzed for indicated cytokine production by Luminex. Data are the mean ± SD (n = 3 independent experiments).$

BDCA3⁺ DCs have a more mature phenotype than BDCA1⁺ DCs. (A–C) Shown are representative plots for BDCA1⁺ DCs (A), BDCA3⁺ DCs (B), and pDCs (C) after isolation (representative of n > 20 donors). (D) Percentage of contaminating cells in isolated DC subsets. Isolated DC fractions were labeled for contaminating cell subsets with antibodies against CD14, CD123, BDCA1, or BDCA3. Live cells were gated on the above markers and the percentage of contaminating subsets was determined. Data are the mean ± SD (n = 7 independent experiments). NA, not applicable (below limit of detection). (E) Phenotype of DC subsets in freshly isolated PBMCs. DCs in PBMCs were labeled for the indicated markers and analyzed by flow cytometry. Isotype background fluorescence was subtracted from the mean fluorescence intensity (MFI), and MFI was normalized to unstimulated BDCA1⁺ DCs. Data are the mean ± SD (n = 6 independent experiments). (F) Phenotype of isolated DC subsets after overnight culture in the presence or absence of TLR7/8 L. After overnight culture, DCs were labeled for the indicated markers and analyzed by flow cytometry. Isotype background fluorescence was subtracted from the MFI, and MFI was normalized to unstimulated BDCA1⁺ DCs. Data are the mean ± SD (n = 6 independent experiments). (G) Endocytic capacity of isolated DC subsets. Overnight cultured isolated DCs were pulsed with 5 µg/ml Alexa Fluor 488 ovalbumin for 20 min at 37 or 4°C, and ovalbumin uptake was analyzed by flow cytometry. Shown is one representative experiment of three. (H) Cytokine production by isolated DC subsets after overnight culture in the presence or absence of TLR7/8 L. Supernatants were collected and analyzed for indicated cytokine production by Luminex. Data are the mean ± SD (n = 3 independent experiments).

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