Cd11b mac 1

BMDM∅ were washed, treated with Fc block (eBiosciences, San Diego, CA), then were incubated with fluorescently labeled antibodies against mouse F4/80 (eBiosciences), MHCII (eBiosciences), TLR2 (eBiosciences), TLR4 (eBiosciences), CD11b (MAC-1; BD Pharmingen, San Jose, CA), CD14 (eBiosciences), CD18 (BD Pharmingen), CD80 (Santa Cruz Biotechnology, Santa Cruz, CA), and CD86 (BD Pharmingen), or isotype control antibodies, per manufacturer’s instructions. The cells were then fixed with 2% buffered paraformaldehyde, 10,000 gated events were collected for each sample and MFI, and percentage of positive staining cells were determined.

The human embryonic stem cell line H1-ESC, H7-ESC, H9-ESC were obtained from WiCell Research Institute (Madison, WI). H1-ESC hES cells were maintained undifferentiated on 6-well plates coated with growth factor-reduced Matrigel (BD Biosciences) in mTeSR1 media (StemCell Technologies) and the media was changed daily. Cells were passaged every three days. To differentiation embryonic stem cells into MSC H1-ESC cells were expanded to 10 cm Matrigel-coated tissue culture dishes. At approximately 80% confluence, cells were dissociated and grown as embryoid bodies which were dissociated and cultured in Mesencult Proliferation Kit (StemCell Technologies).
Primary MSC were isolated from patient bone marrow (UNC IRB exemption 09-0127) using a Ficoll gradient. The mononuclear fraction was isolated and plated. After 3-4 days adherent cells were selected by magnetic depletion according to manufacturer's instructions (MACS) with CD11b/Mac-1 (BD #555387) and CD45 (BD #555481). The negative fraction consisting of MSCs was then plated and expanded for analysis. HKC were passaged twice a week or media changed every 2 days if not confluent.