Fitc conjugated anti human cd44 antibody

Fluorescence-activated cell sorting (FACS) was conducted to analyze the expression of the CD44 and CD24 markers. Briefly, 1 × 10⁶ cells were suspended in 100 μL of immunofluorescence staining buffer supplemented with PE-conjugated anti-human CD24 antibody (BD Pharmingen, San Diego, CA, USA) and FITC-conjugated anti-human CD44 antibody (BD Pharmingen) and incubated for 10 min at 4 °C. Following incubation, the cells were washed with PBS and the CD44⁺/CD24⁻ cell population was determined using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

For analysis of CD44-positive cell surface antigen expression, untreated and HA-treated DPSCs were harvested by trypsinization, washed with PBS, centrifuged into cell pellets and resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.5 % bovine serum albumin). The cells were stained for 30 min at 4 °C with a FITC-conjugated anti-human CD44 antibody (BD Biosciences, San Jose, CA, USA) or an isotype-matched FITC-conjugated IgG control antibody (BD Biosciences). Flow cytometry was performed using an EPICS Altra flow cytometer (Beckman Coulter, Brea, CA, USA) and the data were analyzed using Expo-3 v1.2B software (Beckman Coulter).
For cell cycle analysis, the cell cycle distribution of cells was assayed after 48 h by using flow cytometry to measure the DNA content of nuclei labeled with PI according to the manufacturer’s instructions (BD Pharmingen, BD BioSciences). Data acquisition and analysis were performed using an EC800 flow cytometer (Sony Biotechnology, Tokyo, Japan) with EC800 analysis software (Sony Biotechnology).