Apc conjugated c kit

Monocytes were isolated from total BM cells by Ficoll-Paque-PLUS (Cytiva, Tokyo, Japan) before dead cells were removed with a LIVE/DEAD Fixable Blue DEAD cell stain kit (Thermo Fisher Scientific Inc.). The samples were stained with a Biotin Mouse lineage panel (BD Biosciences, San Jose, CA, USA), followed by PECy7-conjugated streptavidin, APC-conjugated c-kit, and APCCy7-conjugated Sca-1 (all from BD Biosciences), and then LSK cells were isolated by a FACS Fusion (BD Biosciences). An RNeasy Plus Micro kit (Qiagen) was used to extract RNA from LSK cells, and then the samples were sent to Takara Bio Inc. (Shiga, Japan), where the RNA was amplified and microarray analysis was performed.

To monitor the expression of cell surface markers c-Kit, Sca-1, Mac-1, and Gr-1, cells were stained by APC-conjugated c-Kit, PE-cy7-conjugated Sca1, PE-conjugated Mac-1, and percp-cy5.5-conjugated Gr-1 antibodies purchased from BD Biosciences. To monitor apoptosis, cells were stained with PE-conjugated annexin V and 7-AAD using PE Annexin V Apoptosis Detection Kit I from BD Biosciences. Stained cells were evaluated using FACS Canto-II and data were analyzed by FlowJo_V10 software.

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.