S1 Fig. β-glucans does not contain LPS and does not induce hTLR4 mediated NF-κB activation.
(A) 100 μg/mL β-glucans (MacroGard1) was tested for LPS contamination using a
recombinant factor C LAL assay preparation. A 5 EU spiked control was included (n = 1). (B)
Different concentrations (1 mg/mL– 0.1 μg/mL) of commercial β-glucans (MacroGard1) and
LPS (100 pg/mL) were tested for their NF-κB activation via hTLR4 (n = 3).
S2 Fig. Phenotype of cryopreserved cultured porcine mononuclear phagocytes. Gating
strategy following multicolour flow cytometry staining using Abs against CD172a, SLA Class-
II and CD80/86. Cells showing high forward scatter (FSC-A) and side scatter (SSC-A) profiles
were gated, followed by the selection of single cells (FSC-W/H and SSC-W/H) and viable cells
(SSC-A/7-AAD). Among these cells, BMDCs were defined as the CD172a+/high cells (SSC-A/
CD172a) expressing SLA Class-II and CD80/86.
S3 Fig. Phenotype of CD172a+/- (intermediate) cell population. Gating strategy of (A)
frhBMDCs and (B) cryoBMDCs following multicolour flow cytometry staining using Abs
against CD172a, SLA Class-II and CD80/86. The CD172a+/- (intermediate) cell population
(SSC-A/CD172a) does not express SLA Class-II and CD80/86 in both frhBMDC and
cryoBMDC cell cultures.
S4 Fig. FrhBMDCs and cryoBMDCs upregulate SLA Class-II in a dose-dependent manner
upon stimulation with LPS. (A) FrhBMDCs and cryoBMDCs (obtained from the same animal,
n = 1) were stimulated with different concentrations of LPS or unstimulated using cell
culture medium (negative control; Ctrl). After 24 hours, the expression (MFI) of the maturation
markers SLA Class-II were measured using Flow Cytometry. The data are shown as the
means ± the standard error of the mean (SEM) of three technical replicates. A one-way
ANOVA with a Dunnett’s post hoc test was performed, comparing multiple groups to the plots of SLA Class-II expression on LPS stimulated frhBMDCs and cryoBMDCs. The contour
plots are based on forward scatter (y-axis) and SLA Class-II expression (x-axis). The highest
concentration of LPS (10 μg/mL) and cell culture medium (negative control; blue) are presented
in this figure.
S5 Fig. SLA Class-II is not upregulated upon stimulation with EcN, β-glucans or LPS.
Immature (A) frhBMDCs and (B) cryoBMDCs (obtained from the same animal) were stimulated
with different concentrations of E. coli Nissle 1917, β-glucans or LPS. Unstimulated cells
are represented by the white bars (negative control; Ctrl). After 24 hours, the upregulation of
SLA Class-II was measured using Flow Cytometry (n = 4 animals). Relative fold change was
calculated by dividing the MFI of stimulated BMDC/MFI of unstimulated BMDC (Ctrl) of
each animal. The data are shown as the means ± the standard error of the mean (SEM) of 4
animals. A one-way ANOVA with a Dunnett’s post hoc test was performed, comparing multiple
groups to the untreated cells (control): = P<0.001, P<0.01 and P<0.05.