Anti cd44 allophycocyanin apc

Anti-CD45-PeCy7, anti-CD11b-PeCy5, anti-CD166-phycoerythrin (PE), anti-CD105-PE, anti-CD90-PeCy5, anti-CD34-PE, isotype control fluorescein isothiocyanate (FITC) human IgG1, and isotype-control PE human IgG2a were purchased from Beckman Coulter (Indianapolis, IN). Anti-CD44-allophycocyanin (APC) was purchased from BD Biosciences (San Jose, CA). Type 1 collagenase, bovine serum albumin (BSA, fraction V), calcium chloride, cetylpyridinum chloride (CPC) dexamethasone, isobuytlmethylxanthine, indomethacin, ascorbate 2-phosphate, β-glycerol phosphate, alizarin red s, and oil red o were purchased from Sigma (St. Louis, MO).

Two million cells were harvested from 85% confluent flasks and resuspended in PBS with 0.1% BSA. The cells were washed and incubated at 4°C for 30 minutes with anti-CD44-allophycocyanin (APC) (1:20 dilution, clone G44-26, BD Biosciences) and anti-CD133-phycoerythrin (PE) (1:20 dilution, clone AC133, MiltenyiBiotec) antibodies, or mouse-specifc IgG2b ĸ-APC (1:100dilution, BD Biosciences) and IgG1-PE (1:20 dilution, Miltnyi Biotec) antibodies. The cells were then washed and resuspended in PBS with 0.1% BSA and 2 μg/mL propidium iodide (PI), and a C6 FACS (BD Biosciences) was used for all analyses. The cells were first gated on the basis of side-scatter and forward-scatter, followed by the exclusion of nonviable (PI-positive) cells. The CD44⁺ and CD133⁺ gates were created on the basis of cellular staining with the isotype control antibodies (IgG2bĸ-APC and IgG1-PE, respectively).

DLD-1 parental and KOOKi-67 clones were subjected to flow cytometry analysis for the expression of CD44 and CD133. Briefly, 2 million cells were harvested from 85% confluent flasks and resuspended in PBS with 0.1% BSA. The cells were washed and incubated at 4°C for 15 minutes with anti-CD44-allophycocyanin (APC) (1:20 dilution, clone G44-26, BD Biosciences) and anti-CD133-phycoerythrin (PE) (1:20 dilution, clone AC133, Miltenyi Biotec) antibodies, or mouse-specific IgG_2b ĸ-APC (1:100 dilution, BD Biosciences) and IgG₁-PE (1:20 dilution, Miltnyi Biotec) antibodies. The cells were then washed and resuspended in PBS with 0.1% BSA and 2μg/mL propidium iodide (PI), and a FACSCalibur flow cytometer (BD Biosciences) was used for all analyses. The cells were first gated on the basis of side-scatter and forward-scatter, followed by the exclusion of nonviable (PI-positive) cells. The CD44⁺ and CD133+ gates were created on the basis of cellular staining with the isotype control antibodies (IgG_2b ĸ-APC and IgG₁-PE, respectively).