Iate washing, all three cell lines were admixed and resuspended in BD Pharmingen Stain Buffer (BD Biosciences) with the addition of 5 mM EDTA to prevent reaggregation. Cells were plated into 96-well round bottom plates (BD Biosciences) at 30,000 cells (10,000 for each cell line) per well for staining. Cell surface staining was done with antibodies reconstituted with 16PBS at a concentration of 0.5 mg per test and cells were stained live on ice for 20 minutes. Cells were washed thrice with staining buffer and then stained with species-specific Alexa647 secondary antibodies (BD Biosciences) for 20 minutes on ice. Cells were washed three times more in staining buffer, then resuspended in staining buffer with 7AAD (BD Biosciences) for live cell determination. Cells were analyzed on a FACS Canto system (BD Biosciences) equipped with a High Throughput Sampler (with plate loader) and data were compiled using with FlowJo software and a Microsoft Excel 2007 template from BD Biosciences (http://www.bdbiosciences.com/ support/resources/stemcell/index.jsp#stemtools) for generation of heat maps.b2-microglobulin* CD9 CD44 CD46 CD47 CD49b/Integrin a2 CD49f/Integrin a6 CD58/LFA-3 CD59/MIRL CD63 CD71/TFRC CD81 CD97 CD98/LAT1 CD104/Integrin b4 CD146/MCAM CD147/BSG CD151 CD164 CD171/L1CAM CD321/F11 Receptor CD340/Her2 HLA-A,B,C* HLA-A2* MIC A/B*82.95 85.5 87.86 99.59 97.31 89.14 99.44 98.76 99.76 84.62 96.18 98.7 61.51 86.54 92.26 62.15 99.7 69.32 87.3 69.09 65.46 58.32 97.72 89.05 78.Antibody array results showing surface antigens that were expressed on at least 50 of cells in all three colon cancer cell lines analyzed. Arranged in alphanumeric order. *, Antigens common to all nucleated human cells. Abbreviations: lymphocyte function-associated antigen 3, LFA-3; membrane inhibitor of reactive lysis, MIRL; transferrin 18325633 receptor protein 1, TFRC; large neutral amino acid transporter 1; LAT1; melanoma cell adhesion molecule, MCAM; basigin, BSG; L1 cell adhesion molecule, L1CAM; common leukocyte antigen, CLA. doi:10.1371/journal.pone.0053015.tImmunohistochemistryTissues for IHC and IFC were Title Loaded From File obtained through a dedicated tissue procurement team within the Department of Anatomic Benzimidazole (DRB)] in nuclear extracts [11]. Thus, the presence of W049 protein Pathology at Cleveland Clinic. Samples for IHC were fixed in 4 phosphate-buffered formalin and embedded in paraffin wax for sectioning. IHC staining was performed on a Ventana Benchmark XT automated immunostainer utilizing a Ventana Optiview DAB IHC Detection Kit with CC2 antigen retrieval. Primary antibody, polyclonal rabbit anti-ITGA6, (Sigma-Aldrich, HPA012696) was diluted 1:10.Materials and Methods Ethics statementNormal colon and tumor specimens from patients treated at the Cleveland Clinic were obtained according to protocols approved by the Cleveland Clinic Institutional Review Board (IRB 4134), including written informed consent.ImmunofluorescenceFreshly harvested tumor or normal tissue was snap frozen and banked at 280uC. A gastrointestinal pathologist confirmed the histopathology diagnosis of each specimen independently. Normal tissue was obtained from a site distal from 11967625 the primary colon tumor. Fresh frozen tissues were sectioned at a 6 mm thickness. Slides were fixed with 4 paraformaldehyde, air-dried, and stored at 220uC until use. After treatment with 10 normal goat serum and 0.1 Triton X-100 (Sigma-Aldrich) for 45 min, slides were incubated with monoclonal affinity purified mouse anti-human EpCAM (ab20160, Abcam) at a final dilution of 1:200 and monoclonal affinity pur.Iate washing, all three cell lines were admixed and resuspended in BD Pharmingen Stain Buffer (BD Biosciences) with the addition of 5 mM EDTA to prevent reaggregation. Cells were plated into 96-well round bottom plates (BD Biosciences) at 30,000 cells (10,000 for each cell line) per well for staining. Cell surface staining was done with antibodies reconstituted with 16PBS at a concentration of 0.5 mg per test and cells were stained live on ice for 20 minutes. Cells were washed thrice with staining buffer and then stained with species-specific Alexa647 secondary antibodies (BD Biosciences) for 20 minutes on ice. Cells were washed three times more in staining buffer, then resuspended in staining buffer with 7AAD (BD Biosciences) for live cell determination. Cells were analyzed on a FACS Canto system (BD Biosciences) equipped with a High Throughput Sampler (with plate loader) and data were compiled using with FlowJo software and a Microsoft Excel 2007 template from BD Biosciences (http://www.bdbiosciences.com/ support/resources/stemcell/index.jsp#stemtools) for generation of heat maps.b2-microglobulin* CD9 CD44 CD46 CD47 CD49b/Integrin a2 CD49f/Integrin a6 CD58/LFA-3 CD59/MIRL CD63 CD71/TFRC CD81 CD97 CD98/LAT1 CD104/Integrin b4 CD146/MCAM CD147/BSG CD151 CD164 CD171/L1CAM CD321/F11 Receptor CD340/Her2 HLA-A,B,C* HLA-A2* MIC A/B*82.95 85.5 87.86 99.59 97.31 89.14 99.44 98.76 99.76 84.62 96.18 98.7 61.51 86.54 92.26 62.15 99.7 69.32 87.3 69.09 65.46 58.32 97.72 89.05 78.Antibody array results showing surface antigens that were expressed on at least 50 of cells in all three colon cancer cell lines analyzed. Arranged in alphanumeric order. *, Antigens common to all nucleated human cells. Abbreviations: lymphocyte function-associated antigen 3, LFA-3; membrane inhibitor of reactive lysis, MIRL; transferrin 18325633 receptor protein 1, TFRC; large neutral amino acid transporter 1; LAT1; melanoma cell adhesion molecule, MCAM; basigin, BSG; L1 cell adhesion molecule, L1CAM; common leukocyte antigen, CLA. doi:10.1371/journal.pone.0053015.tImmunohistochemistryTissues for IHC and IFC were obtained through a dedicated tissue procurement team within the Department of Anatomic Pathology at Cleveland Clinic. Samples for IHC were fixed in 4 phosphate-buffered formalin and embedded in paraffin wax for sectioning. IHC staining was performed on a Ventana Benchmark XT automated immunostainer utilizing a Ventana Optiview DAB IHC Detection Kit with CC2 antigen retrieval. Primary antibody, polyclonal rabbit anti-ITGA6, (Sigma-Aldrich, HPA012696) was diluted 1:10.Materials and Methods Ethics statementNormal colon and tumor specimens from patients treated at the Cleveland Clinic were obtained according to protocols approved by the Cleveland Clinic Institutional Review Board (IRB 4134), including written informed consent.ImmunofluorescenceFreshly harvested tumor or normal tissue was snap frozen and banked at 280uC. A gastrointestinal pathologist confirmed the histopathology diagnosis of each specimen independently. Normal tissue was obtained from a site distal from 11967625 the primary colon tumor. Fresh frozen tissues were sectioned at a 6 mm thickness. Slides were fixed with 4 paraformaldehyde, air-dried, and stored at 220uC until use. After treatment with 10 normal goat serum and 0.1 Triton X-100 (Sigma-Aldrich) for 45 min, slides were incubated with monoclonal affinity purified mouse anti-human EpCAM (ab20160, Abcam) at a final dilution of 1:200 and monoclonal affinity pur.
