Cd184 apc

MOLM-16 and OCI-AML3 cells were treated for 6 h with AZD1208 (2 μM), AZD2014 (1 μM), or the combination. Cells were then fixed with 1.6% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and subjected to permeabilization in ice-cold methanol (70% in PBS; 1 mL/million cells) for 20 min. After washing twice, cells were resuspended in 1% bovine serum albumin in PBS. Antibodies were added to the cell suspension and incubated for 30 min. Antibodies used were Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) mouse monoclonal antibody (Alexa Fluor 488 Conjugate #4374; Cell Signaling Technology, Beverly, MA); Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP rabbit monoclonal antibody (Alexa Fluor 647 Conjugate #4851; Cell Signaling Technology); Phospho-Akt (Ser473) rabbit monoclonal antibody (Alexa Fluor 647 Conjugate #A88881; Beckman Coulter, Inc., Brea, CA); and CXCR4 (CD184-APC, #555976; BD Biosciences). After washing twice, cells were resuspended and analyzed by a Gallios flow cytometer (Beckman-Coulter).

hESC-derived neural cultures were washed with PBS, trypsinized with 0.05% trypsin for 1–2 min at room temperature and resuspended into single cells using DMEM/F12 with Glutamax containing 10% FBS. For cell surface staining, cells were incubated with CD184-APC (560936; BD Pharmingen), CD24-PE-Cy7 (561646; BD Pharmingen), CD44-PE (51-9007231; BD Pharmingen) and/or CD271-Alexa fluor 647 or BV421 (560877 or 562562; BD Pharmingen) antibodies in PBS containing 1% bovine serum albumen (BSA) according to manufacturer’s protocol. Stained and unstained cells were analyzed using either a BD LSR-II or LSRFortessa flow cytometry machine (BD Biosciences). Control hES9 hESCs or hESC-derived NPCs were used for mCherry control and IgG-PE-Cy7, IgM-FITC, IgG-BV421 and IgG-APC (BD Pharmingen) were used for isotype controls. Cell sorting was performed using either BD FACSAria II or BD Influx cell sorter. Gating for all sorts was defined by isotype control staining. Flow cytometry data analysis was performed using FlowJo software (Tree Star, Inc.).

Differentiated PSCs were digested into single cells by TrypLE (Gibco, #12604021) and washed twice with DPBS containing 2% FBS. Cells were then incubated with CD184-APC (BD, #555976) for 30 min. As for the following intracellular flow cytometry, cells were fixed according to the manufacturer's instructions of Transcription Factor Buffer Set (BD, #562574), and then incubated with SOX17-Alexa488 (BD, #562205) antibody. Corresponding isotype was used as control. The SOX17⁺ or CXCR4⁺ cells were detected by FACSCelesta flow cytometer (BD) or CytoFLEX flow cytometer (Beckman Coulter) and analyzed by FlowJo software.