7 aad negative

The livers were perfused through the portal vein with fluorescence-activated cell sorting (FACS) buffer [1 g bovine serum albumin (BSA) in 500 mL PBS] and then minced well. The filtrate was centrifuged at 50 g for 1 min, and the supernatant was washed once. The cells were suspended in 25% Percoll and overlaid in 50% Percoll to distinguish resident macrophages from monocytes. After centrifugation at 2000 rpm for 20 min, the cells were collected from the middle layer and were washed and resuspended in an RPMI1640 medium. Flow cytometry was performed as described previously [18 (link)]. Briefly, the cells were collected from the upper phase of a Percoll gradient. After blocking with anti-FcR (CD16/32, BD bioscience, NJ) for 20 min, the cells were incubated with specific monoclonal antibodies at 4 °C for 30 min. The liver mononuclear cells were gated as 7-AAD negative CD45.2-FITC (BD bioscience) positive cells. Liver macrophages were stained with PE-conjugated anti-mouse F4/80 mAb and antigen-presenting cell (APC)-conjugated anti-mouse CD11b mAb (both from BD biosciences). Background fluorescence was assessed by staining with the relevant isotype control Abs. Stained cells were analyzed by flow cytometry (FACS Cant II, Becton Dickinson Co. Franklin Lakes, NJ), and data were analyzed using the FlowJo software (FlowJo, LLC, Ashland, OR).

For undifferentiated hESC cultures, cells were dissociated with Cell Dissociation Buffer (Life Technologies) for 5–10 minutes. Dissociated single cells were incubated with SSEA3 (rat anti-human IgM, Hybridoma Bank) or A2B5 (mouse anti-human IgM, Chemicon) antibodies for 40 minutes and then identified with fluorescent-conjugated secondary antibodies (Alexa 647-conjugated goat anti-rat IgM or Alexa 647-conjugated goat anti-mouse IgM, BD Biosciences). Anti-Oct4 (BD Biosciences) staining was identified using Alexa 647-conjugated goat anti-mouse IgG (BD Biosciences). Additionally, APC-conjugated c-kit (BD Biosciences) was also used for flow. Hematopoietic or neural cells derived from day 20 EBs were detected using CD45 (BD Biosciences), nestin (R&D Systems) antibodies, respectively. Following each staining, live cells were distinguished as 7-AAD negative (BD Biosciences). Flow analysis was performed on a FACS Calibur running Cell Quest Software (BD Biosciences). Postrun analysis was performed using FlowJo version 8.5.3 (Treestar) http://www.flowjo.com/ Ashland, OR, USA.