Journal: Frontiers in Immunology
Article Title: Anaphylatoxin C5a Regulates 6-Sulfo-LacNAc Dendritic Cell Function in Human through Crosstalk with Toll-Like Receptor-Induced CREB Signaling
doi: 10.3389/fimmu.2017.00818
Figure Lengend Snippet: C5a affects the pro-inflammatory potential of 6-sulfo LacNAc dendritic cells (slanDCs). (A) Slan, CD123, CD1c, CD141, CD11c, HLA-DR, and CD14 expressions in the 120 different clusters defined by spanning-tree progression analysis of density-normalized events (SPADE). (B) C5aR expression in the 120 clusters defined by SPADE. Different dendritic cell (DC) subsets are indicated with black ellipses and were selected based on the expression of known DC identification markers ( , ). pDCs were selected as CD123+, CD11c−, CD1c−, CD14−, and slan−; slanDCs were CD11c+, CD1c−, CD14−, slan+, and CD123−; MDC1s were CD11c+, CD1c+, CD14−, slan−, and CD123−; and MDC2s were CD11c+, CD141++, CD1c−, CD14−, and slan−. (A,B) Fluorescent intensity of marker expression is visualized using a color scale. (C) Sorted slanDCs were stimulated overnight with LPS or R848 or left untreated. The production of TNF-α and IL-12p40 in supernatants is depicted ( n = 4). (D) The production of TNF-α and IL-12p40 by slanDC determined after overnight stimulation with R848 in the absence or presence of C5a ( n = 5). Cytokine production was measured using ELISA.
Article Snippet: The following kit or antibodies were used to stain immune cells: anti-HLA-DR brilliant ultraviolet (BUV) 395 (cat# 564040), anti-CD3 brilliant violet (BV) 510 (cat# 563109), anti-CD3 phycoerythrin (PE) (cat# 345765), anti-CD11c PE (cat# 347637, RRID:AB_2129929), anti-CD14 allophycocyanin (APC) (cat# 345787), anti-CD19 BV510 (cat# 562947), anti-CD56 BV510 (cat# 563041), anti-CD123 BV650 (cat# 563405), anti-pP38 (pT180/pY182) Alexafluor (AF) 647 (cat# 612595, RRID:AB_399878), and anti-pERK1/2 (ERK1 pT202/pY204; ERK2 pT184/pY186) AF488 (cat# 612592), which were obtained from BD Biosciences (San Jose, CA, USA).
Techniques: Expressing, Marker, Enzyme-linked Immunosorbent Assay