Alkaline phosphatase detection kit scr004

Cells were disassociated using Versene (EDTA; Lonza), washed with phosphate‐buffered saline (PBS), and fixed in paraformaldehyde (2% final concentration in PBS) at 37°C for 10 minutes. After washing with PBS, the cells were permeabilized with prechilled methanol (−20°C) and incubated at 4°C for 30 minutes, followed by a washing step. Cells (0.2–0.5 × 10⁶) were resuspended in a total volume of 200 μl PBS containing 1% bovine serum albumin and incubated with appropriate amounts of anti‐CD44‐BV421 (Catalog number 562890; BD Biosciences, Oxford, UK; 1:300 dilution) and anti‐TRA‐1‐60‐FITC (Catalog number FCMAB115F; Merck Millipore; 1:100 dilution) monoclonal antibodies for 1 hour on a shaker, plate in the dark at room temperature. Finally, samples were washed using BD FACS Lyse Wash Assistant (BD Biosciences) and immediately analyzed on a flow cytometer. Fluorescence‐activated cell sorting (FACS) analysis was performed using BD FACS Canto II flow cytometer with FACSDiva software (BD Biosciences). A minimum of 20,000 events were recorded for each sample. Fluorescence minus one control (for each antibody) was used to gate the subpopulations.
Alkaline phosphatase detection was performed with alkaline phosphatase detection kit (SCR004; Millipore) according to manufacture instructions.

iPS cell colonies were cultured on MEF feeder layers for 5 days. Cells were then analyzed for AP activity using an Alkaline Phosphatase Detection Kit (SCR004; Millipore, Billerica, MA) according to the manufacturer's protocol.