Immune cells isolation and culture
PMɸ were isolated from male FVB WT mice as previously
described (Ray A et al., 2010), but with some modifications. A
euthanized mouse fully submerged in 75% ethanol was fixed on its back,
and the inner skin lining exposed with scissors and forceps. 8-10 ml of
37°C PBS containing 3% fetal bovine serum (FBS) was carefully injected
into the peritoneal cavity, and the peritoneum was massaged for 5 min.
The cell suspension was then collected from the peritoneum, centrifuged
at 1500 rpm for 10 min. The pellet of cells was resuspended in DMEM high
glucose (Gbico™; Thermo Fisher Scientific) containing 10% FBS and
cultured for four days. After trypsinization, cells were washed twice
with PBS and incubated for 30 min with the following monoclonal
antibodies: APC anti-mouse F4/80 (123116; BioLegend), FITC
anti-mouse/human CD11b (101206; BioLegend) for macrophages
identification and characterization using flow cytometry (LSRFortessa;
BD Biosciences).
For the ALX treatment group, cultured PMɸ were
pre-treated for an hour with 35μM/ml of ALX and challenged with 10μM/ml
of ISO and, or 1μg/ml LPS. Next, treatments with 35μM/ml of ALX and, or
10μM/ml FSK followed. The treated PMɸ were incubated at
37°C and 5% CO2 for 24 h. The supernatants from the
culture were collected and stored in -80°C for cytokine and cAMP assays.