Pe conjugated anti mouse igg

Recombinant human VCAM-1/CD106 Protein and Recombinant Human/Rhesus Macaque/Feline CXCL12/SDF1α were from R&D Systems (Minneapolis, MN, USA). Human fibronectin, doxorubicin and FAK inhibitor PF-228 were purchased from Millipore Sigma (Oakville, ON, Canada). The PYK2 inhibitor VS-6063 was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Anti-phospho-PYK2 (Tyr-402) and anti-PYK2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The fluorescein isothiocyanate (FITC)-conjugated Annexin V, Phycoerythrin (PE)-conjugated anti-human α4 integrin, Alexa 647-conjugated anti-human α5 integrin antibodies, FITC-conjugated anti-human CD3 and PE-conjugated anti-mouse IgG were purchased from BD Biosciences (San Jose, CA, USA). Anti-human β1 integrin (4B4) was purchased from Beckman Coulter Life Sciences (Mississauga, ON, Canada).

For live cell analysis, dispersed ES cells were washed with PBS and suspended with FACS buffer (PBS/2% fetal bovine serum/0.02% NaN₃), and the EmGFP signals were analyzed by FACSCantoII or LSRFortessa X-20 (Beckton Dickinson) at FITC channel. For immunostaining analysis, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with FACS buffer containing 0.5% Tween 20 were incubated with anti-H3K27ac or anti-FLAG M2, and with anti-GFP antibodies at 4 °C for overnight. The cells were washed with FACS buffer containing 0.1% Tween 20 three times followed by incubation with secondary antibodies, PE-conjugated anti-mouse IgG (BD Pharmingen), and Alexa Fluor 488-conjugated anti-rat IgG (Invitrogen), for 30 min. Washed samples were analyzed by LSRFortessa X-20 (Beckton Dickinson). Data was analyzed by using BD FACSDiva software (Becton Dickinson). Each analysis was done in two to five independent experiments with biological triplicates for each set of experiment.