Supplementary Information

Transcription

1 Supplementary Information Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates Lars Franken, arika Klein, arina Spasova, nna Elsukova, Ulf Wiedwald, eike Welz, ercy Knolle, ichael Farle, ndreas Limmer and Christian Kurts

2 Supplementary Figure 1: FITC C E-Cy7 unstained unstained DCs Supplementary Figure 1: Flow cytometrical analysis of the autofluorescent properties of and DCs. The autofluorescence was analyzed in the indicated channel. The signal is depicted as a black line, the DC signal as grey background.

3 Supplementary Figure 2: ml) 5 3 Source cells LN ml) Dendritic cells T cells 15 5 ml) cells Dendritic cells T cells ml) 3 ml) 5 3 ml) 3 Supplementary figure 2: -contaminations alter cytokine concentrations in supernatants from in vitro cultures of conventional dendritic cells, T Cells and -Cells. cells, T cells and DCs were obtained by magnetic cell purification from spleens or the mesenteric lymph nodes without or with -depletion using the 3 antibody method. Cells were activated as described and 18 hours later supernatants were collected and cytokine levels were analyzed. () TNF-alpha concentrations in supernatants of cells, DCs and T cell cultures. () CCL3-concentrations in supernatants of - or T cell cultures or CCL4-concentrations in supernatants of DC cultures. esults are shown for one representative of two to three individual experiments using 3 mice per group. Error bars, s.d. (n=3 mice); <.5; <.1

4 Supplementary Figure 3: undepleted 3-antibody protocol CFSE proliferation of OT-II-T-cells in % no OV undepleted 3 antibody protocol Supplementary Figure 3: ntigen presentation by CD11c cells and obtained by CS. The cells were purified by the standard protocol or depleted of using our 3-antibody protocol. () Flow cytometrical analysis of the proliferation of CFSE-labeled OT-II T cells. Cells incubated with ovalbumine are depicted as black line, negative control that did not receive ovalbumin as grey background. () Statistical evaluation of ().

5 Supplementary Figure 4: 15 CD163 Expression level relative to Spleen 6 (%) 5... Source Spl Spl Spl LN Spl Spl Spl LN Spl Spl Spl LN Spi-C expression cells Dendritic cells T cells Supplementary Figure 4: -contaminations are responsible for CD163 mn expression in splenic DCs, T cells or cells isolated by conventional CS. mn levels of CD163 determined by T-C., source tissue, SpiC-expression and -depletion as indicated. esults are shown for one representative of two individual experiments (n=3); mean ± s.e.m.; p<.5, p<.1 (One-way nova in combination with a onferroni multiple-comparison test).

6 Supplementary Figure 5: relative CD11c 3 Spi-C expression T cells Spi-C expression T cells Spi-C expression DCs C relative Source Spi-C expression Spl Spl Spl LN Spl cells relative CD Spi-C expression cells relative DEC5 3 Spi-C expression DCs Supplementary Figure 5: -depletion does not reduce the levels of non- genes during T-C analysis. (-C) cells, T cells and DCs were obtained by magnetic cell purification from spleens or the mesenteric lymph nodes without or with -depletion using the 3-antibody protocol. The expression levels of the genes CD8β, CD3ε (), CD11c, DEC5 (), 2 and CD19 (C) relative to GDH was determined using qt-c. s negative control expression of these genes was also determined in purified by magnetic cell separation. esults are shown for one representative of two to three individual experiments using 2-4 mice per group. Error bars, s.d. (n=3 mice); <.5; <.1

7 Supplementary Figure 6: 15 1 relative Hmox1 expression relative Fpna1 expression 1 5 relative VC1 expression undepleted 3-antibody protocol undepleted 3-antibody protocol undepleted 3-antibody protocol undepleted CD11c cells CD11c cells depleted with 3-antibody protocol Isoype staining VC Supplementary figure 6: Genes involved in iron recycling can be detected in CS purificates of CD11c cells. () T-C analysis of CD11c cells and obtained by CS. The cells were purified by the standard protocol or depleted of using our 3-antibody protocol. Depicted are the relative expression levels of Hmox1, Fpn1 and Vcam1. () Flow cytometric analysis of the VC1 expression in cells purified like in (). CD11c cells obtained by the standard CS protocol are depicted as red line, CD11c cells depleted of using our 3-antibody protocol are depicted as black line. Isotype staining depicted as grey background.

8 Supplementary Figure 7: 25K Initial Gating 25K Exclusion of duplets 25K CD45 Gating K K K 15K 15K 15K K.4 K 92.3 K K 5K 5K SSC- 5K K15K K 25K FSC- Life/dead Gating 25K K 15K K FSC- 5K K15K K 25K FSC-W C Gating FSC CD45 Standard L/D gating excludes autofluorescent 5K Correct gating displays autofluorescent F4/ Life/Dead F4/ CD11c Supplementary Figure 7: Gating scheme employed for flow cytometrical analysis of splenocytes and CS isolates. () Depicted is the initial gating to exclude small cells fragments (SSC- versus FSC-C, left dot plot), cell duplets (FSC- versus FSC-W, middle dot plot) and the gating on CD45 cells (FSC- versus CD45, right dot plot). () Gating scheme for the exclusion of dead cells and from flow cytometrical analysis. Dead cells are excluded by staining with a dead cell detection dye. are either left in the viable cell population (upper dot plot) or are excluded as shown (lower dot plot). The dot plots on the right demonstrate the composition of the viable cell population.

9 Supplementary Table 1: rimers used for T-C Gene Sense ntisense GITL CCTCGCCCTGCCTC CCGGTCCTTGGCCGT γ TGTGGCCCGCCTGC GTGTCCCTGGTTTTCGT 2 CCCTCCCCGTGTGCT TCGCTTGGCTGCTGTGT CD19 CCTGCCTCGGGGC CTTTGGTCTCCTGGCGG CD3ε CCTCCTGCTGTTGGCCTT GTGCCCTGTCCTCGCT CD8β CTGCCCCCCGGCT TCTCCTCCGCCCGT DEC5 GGCCTGTCTCGCTTCCT GTCCGGGCTCGGGTTT CD163 CCGGCCTCTTGGTTTGTG GGTTTTCCGGGTTTCGC CD11c TTCCTGGCTGTTGGCTTGTG GGCTGTGCTCCCGGC CGCTCGGCGGTGGCTCTG GCTTGCGCTTGCCCTTGCC Hmox1 GCTTTGCTGGTGTGGCT GTGGGCCCTCCGG Fpn1 CCCTGCTCTGGCTGTG GGTGGGCTCTTGTTCCTT Vcam1 TTTTCTGGGGCGGGTT CGTCGCCCGTCC