Figure 6. Dendritic cell staining and sorting with bCCL19-SAPE.

(A) Immature DCs were grown from CD14⁺ fractions of buffy coats and matured with PolyI:C/PGE₂ for 48 hours. Cells were stained as in Figure 1. (i) Immature DCs. (ii) Mature DCs. (iii) Maturation of human monocyte-derived DC (MoDC) with PolyI:C and PGE2 increases the percentage of cells staining positive with bCCL19-SAPE (n=6). bCCL19-SAPE⁺ gates were set using SAPE controls where <1% of cells showed positivity for SAPE. (B) bCCL19-SAPE staining of murine BMDCs. Immature DCs were grown from murine BM in the presence of GM-CSF and matured with LPS and TNF-α for 24 hours. Cells were stained using 250ng/mL bCCL19 conjugated to SAPE. (i) Immature DCs (ii) DCs matured with LPS/TNF-α. (iii) Percentage of cells staining positive with bCCL19 (n = 7). Gates were set as in (A). (C) Initial sorting of DCs. The percentage of cells staining positive with bCCL19-SAPE is increased from the presort sample (i) to the post-sort sort fraction (ii) leaving behind a negative sort fraction depleted for bCCL19⁺ cells (iii). (D) Quantitative data from comparative DC sorts. Sorting was initially performed with a single LS column. The sort was then performed using LS columns fitted with 23G needles (LS23G, dark grey bars), to slow the flow of cells through the column. After the first sort, the negative fraction of the sample was passed through a second LS23G (LS23G/LS23G, black bars), to increase the yield of positive cells from the sort. Statistics were calculated using 2-way repeated measures ANOVA with Bonferroni post-test. n=3-4, *p<0.05, ***p<0.001.