L (CSC) markers. Notably, recent evidence has suggested that metastases are colonized by these CSCs that possess the functional abilities of self-renewal and multi-lineage differentiation [18?0]. CSCs may also function within tumors to propagate and/or maintain tumors over time and in response totreatment. Multiple groups have proposed various intracellular and extracellular identifying markers for CSCs that often have immunophenotypic similarities to normal tissue stem cells. Importantly, the detection of CSCs can be contingent upon antibody binding of post-translational (e.g. glycosylated) epitopes. The addition of such moieties to peptides often disconnects transcript levels from the amount detected by antibodies (e.g. Prominin-1/CD133 transcripts and the CD133 (AC133) epitope in colon cancer CSCs [21]). Currently studied CSC surface proteins in colon cancer include EpCAMhigh, CD133, CD26, CD166, and CD44, Elesclomol independently or in combination [18,20,22?5]. CD26, a proposed marker of metastatic stem cells [20], and CD166 [24] were included in the antibody array and the results are provided in Figure S1. We performed standard multicolor flow cytometry for EpCAM, CD133, and CD44 to determine their expression individually as well as double and triple staining (Table 4 and Figure S6). Although we did not test the functional stem cell properties of any tumor cell populations, we were able to detect the varied expression of stem cell immunophenotype markers ranging from near absent to complete labeling. These results indicate that stem cell marker immunophenotypes can diverge greatly between tumors and is not always restricted to rare populations. Further characterization of these populations, beyond the scope of the current study, can determine the relevance of these expression patterns to functional phenotypes.Multiplexed FACS Antibody Array in Colon Genz 99067 web CancerTable 1. Broadly expressed tumor antigens.AntigenCell line ( Positivity) SW480 SW620 96.8 83.27 80.57 99.25 98.01 96.74 99.26 98.43 99.66 97.03 93.02 98.67 94.2 85.68 97.66 64.14 99.74 87.31 90.86 83.99 93.87 76.38 99.07 98.37 68.36 HCT116 95.2 79.3 99.6 99.39 99.75 99.66 99.47 99.25 99.42 99.0 99.66 99.36 56.09 81.19 99.49 91.83 100.0 85.71 98.14 58.07 95.79 93.9 99.56 99.21 96.37uC). The Papain dissociation kit was obtained from Worthington Biochemical. Viability of cells was checked using Trypan blue exclusion and found to be .98 . All cells were grown and processed in parallel.High throughput flow cytometry analysisHigh throughout flow cytometry analysis was performed on the three cell lines described above using an antibody screening method developed by BD Biosciences [27]. Antibody screening was done using the BD Lyoplate human cell surface marker screening panel (560747) containing lyophilized antibodies in a 96-well plate format at 0.5 mg/ well. For flow 15826876 cytometry analysis, the cell suspensions were treated with DNAse (in 1 ml PBS with Ca2+, Mg2+, 100 units/ml, 10 ml 1527786 DNAse stock) for 15 minutes at room temperature. Prior to antibody staining, cell lines were barcoded with different viability dyes for simultaneous analysis [28]. SW480 cells were labeled with Horizon Violet Proliferation Dye 450 (BD Biosciences 562158) as per the manufacturer’s protocol at 1 mM. HCT116 was labeled with CFSE (Invitrogen, C34554) as per the manufacturer’s protocol at 1 mM. SW620 was left unlabeled and detected on the basis of being VPD450 and CFSE negative. Efficiency of labeling was .99 . After appropr.L (CSC) markers. Notably, recent evidence has suggested that metastases are colonized by these CSCs that possess the functional abilities of self-renewal and multi-lineage differentiation [18?0]. CSCs may also function within tumors to propagate and/or maintain tumors over time and in response totreatment. Multiple groups have proposed various intracellular and extracellular identifying markers for CSCs that often have immunophenotypic similarities to normal tissue stem cells. Importantly, the detection of CSCs can be contingent upon antibody binding of post-translational (e.g. glycosylated) epitopes. The addition of such moieties to peptides often disconnects transcript levels from the amount detected by antibodies (e.g. Prominin-1/CD133 transcripts and the CD133 (AC133) epitope in colon cancer CSCs [21]). Currently studied CSC surface proteins in colon cancer include EpCAMhigh, CD133, CD26, CD166, and CD44, independently or in combination [18,20,22?5]. CD26, a proposed marker of metastatic stem cells [20], and CD166 [24] were included in the antibody array and the results are provided in Figure S1. We performed standard multicolor flow cytometry for EpCAM, CD133, and CD44 to determine their expression individually as well as double and triple staining (Table 4 and Figure S6). Although we did not test the functional stem cell properties of any tumor cell populations, we were able to detect the varied expression of stem cell immunophenotype markers ranging from near absent to complete labeling. These results indicate that stem cell marker immunophenotypes can diverge greatly between tumors and is not always restricted to rare populations. Further characterization of these populations, beyond the scope of the current study, can determine the relevance of these expression patterns to functional phenotypes.Multiplexed FACS Antibody Array in Colon CancerTable 1. Broadly expressed tumor antigens.AntigenCell line ( Positivity) SW480 SW620 96.8 83.27 80.57 99.25 98.01 96.74 99.26 98.43 99.66 97.03 93.02 98.67 94.2 85.68 97.66 64.14 99.74 87.31 90.86 83.99 93.87 76.38 99.07 98.37 68.36 HCT116 95.2 79.3 99.6 99.39 99.75 99.66 99.47 99.25 99.42 99.0 99.66 99.36 56.09 81.19 99.49 91.83 100.0 85.71 98.14 58.07 95.79 93.9 99.56 99.21 96.37uC). The Papain dissociation kit was obtained from Worthington Biochemical. Viability of cells was checked using Trypan blue exclusion and found to be .98 . All cells were grown and processed in parallel.High throughput flow cytometry analysisHigh throughout flow cytometry analysis was performed on the three cell lines described above using an antibody screening method developed by BD Biosciences [27]. Antibody screening was done using the BD Lyoplate human cell surface marker screening panel (560747) containing lyophilized antibodies in a 96-well plate format at 0.5 mg/ well. For flow 15826876 cytometry analysis, the cell suspensions were treated with DNAse (in 1 ml PBS with Ca2+, Mg2+, 100 units/ml, 10 ml 1527786 DNAse stock) for 15 minutes at room temperature. Prior to antibody staining, cell lines were barcoded with different viability dyes for simultaneous analysis [28]. SW480 cells were labeled with Horizon Violet Proliferation Dye 450 (BD Biosciences 562158) as per the manufacturer’s protocol at 1 mM. HCT116 was labeled with CFSE (Invitrogen, C34554) as per the manufacturer’s protocol at 1 mM. SW620 was left unlabeled and detected on the basis of being VPD450 and CFSE negative. Efficiency of labeling was .99 . After appropr.
