Journal: Clinical and Molecular Allergy : CMA

Article Title: Differential uptake of three clinically relevant allergens by human plasmacytoid dendritic cells

doi: 10.1186/s12948-021-00163-8

Figure Lengend Snippet: Regulation of allergen uptake of pDC varies among different allergens. PDC isolated from fresh peripheral blood or buffy coats by BDCA-4 positive magnetic bead selection were cultured overnight in AIM-V medium containing 10 ng/ml IL-3 in the presence or absence of CpG ODN 2006 (2,5 µg/ml), IL-4 (500 U/ml) or IL-13 (100 U/ml) or allergen as indicated. Prior to incubation, allergens were labeled using an Alexa Fluor 555 staining kit as described in Material and Methods. Allergen concentrations were 20 µg/ml for Der p 1 and 10 µg/ml for OVA and Api. After incubation, surface staining was performed with FITC conjugated Lin-1 and PerCP Cy5.5 conjugated CD123 and allergen uptake was analyzed by flow cytometry. PDC were defined as Lin-1 − /CD123 ++ cells with pDC comprising 24–50%. A Dot plots show examples of allergen uptake regulation, which are summarized in ( B – D ), where box plots show percentages of allergen-positive pDC, central horizontal lines indicate medians, box borders represent IQR, whiskers indicate minima and maxima. B n = 6, C n = 5, D n = 8 experiments. Significance levels were assessed by Friedman test with p-values: ** p < 0.005 and * p < 0.05

Article Snippet: The TLR9 agonist CpG oligodeoxynucleotide ODN 2006 (CpG B) with the specific sequence 5’-TCG TCG TTT TGT CGT TTT GTC GTT-3’ and the cytokines IL-3, IL-4 and IL-13 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Isolation, Selection, Cell Culture, Incubation, Labeling, Staining, Flow Cytometry

Journal: Clinical and Molecular Allergy : CMA

Article Title: Differential uptake of three clinically relevant allergens by human plasmacytoid dendritic cells

doi: 10.1186/s12948-021-00163-8

Figure Lengend Snippet: ODN 2006 suppresses uptake of Der p 1, but not Api in healthy and allergic subjects. PBMC from fresh blood samples were enriched for panDC and cultured overnight in AIMV medium containing 20 ng/ml IL-3 in the presence or absence of CpG ODN 2006 (2,5 µg/ml), IL-4 (500 U/ml) or IL-13 (100 U/ml) or allergen as indicated. Prior to incubation allergens were labeled using an Alexa Fluor 555 staining kit as described. Allergen concentrations were 20 µg/ml for Der p 1 and 10 µg/ml for bee venom. After incubation, allergen uptake was analyzed using flow cytometry. Frequencies of pDCs in the DC-enriched samples reached 24–50% as defined by Lin-1 − /CD123 ++ staining. A Results from healthy controls without house dust mite (HDM) allergy (white boxes, n = 6) and individuals allergic to HDM (grey boxes, n = 5) are shown. B Data from healthy controls without bee allergy (white boxes, n = 8) and individuals allergic to bee venom (grey boxes, n = 12) are shown. Box plots illustrate percentages of allergen-positive pDC, central horizontal lines indicate medians, box borders represent IQR, whiskers indicate minima and maxima. Significance levels were **p < 0.005 and *p < 0.05, assessed by Friedman test, followed by Dunn’s multiple comparisons test

Article Snippet: The TLR9 agonist CpG oligodeoxynucleotide ODN 2006 (CpG B) with the specific sequence 5’-TCG TCG TTT TGT CGT TTT GTC GTT-3’ and the cytokines IL-3, IL-4 and IL-13 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Cell Culture, Incubation, Labeling, Staining, Flow Cytometry