Surface markers (CD45, EpCAM (CD326), integrins β3, β4, and αVβ5) were stained in the first step, intracellular staining was performed during the second step. Samples were incubated at RT for 10 min with 5 μL of Fc Receptor Blocking Solution (Human TruStain FcX, Sony Biotechnology, San Jose, CA, USA). Next, monoclonal antibodies were added and incubated at RT for 20 min: APC-Cy7-anti-CD45 (clone HI30, IgG1, Sony Biotechnology, USA), BV 650-anti-EpCAM (clone 9C4, IgG2b, Sony Biotechnology, USA), BV 421-anti-β3 integrin (clone VI-PL2, BD Biosciences, USA), Alexa Fluor 488-anti-β4 integrin (clone 422325, R&D Systems, Minneapolis, MN, USA), BV Alexa Fluor 750-anti-αVβ5 integrin (clone P5H9, R&D Systems, USA). The unstained control and antibody quality control were performed. The appropriate isotype antibodies were added to the isotype control sample at the same concentration. After incubation, red blood cells were lysed by 250 μL OptiLyse C buffer (Beckman Coulter, Villepinte, France) at RT for 10 min in dark and washed in 1mL Cell Wash buffer (BD Biosciences, USA) at 800× g for 6 min.
For intracellular staining, cells were permeabilized by 250 μL BD Cytofix/Cytoperm (BD Biosciences, USA) at 4 °C for 30 min in the dark and washed twice in 1mL BD Perm/Wash buffer (BD Biosciences, USA) at 800× g for 6 min. Then, samples were diluted in 50 μL BD Perm/Wash buffer (BD Biosciences, USA) and incubated at 4 °C for 10 min in the dark with 5 μL of Fc Receptor Blocking Solution (Human TruStain FcX, Sony Biotechnology, USA). Next, monoclonal antibodies BV 650-anti-EpCAM (clone 9C4, IgG2b, Sony Biotechnology, USA) were added and incubated at 4 °C for 20 min. The appropriate isotype control antibodies at the same concentration were added to the control sample. After incubation, samples were washed in 1mL Cell Wash buffer (BD Biosciences, USA) at 800× g for 6 min. Then, samples were diluted in 100 μL Stain buffer (Sony Biotechnology, USA). Compensation beads (VersaComp Antibody Capture Bead kit, Beckman Coulter, USA) were used for compensation control. The immunofluorescence was analyzed on the Novocyte 3000 (ACEA Biosciences, San Diego, CA, USA).
The gating strategy was as follows: using forward (FSC) and side scatter (SSC), debris were descriminated, and doublets were also discriminated by plotting FSC area vs. FSC height. Further analysis included only CD45-negative and EpCAM-positive cells. Then, we characterized CTCs by the expression of integrins β3, β4, and αVβ5.
For intracellular staining, cells were permeabilized by 250 μL BD Cytofix/Cytoperm (BD Biosciences, USA) at 4 °C for 30 min in the dark and washed twice in 1mL BD Perm/Wash buffer (BD Biosciences, USA) at 800× g for 6 min. Then, samples were diluted in 50 μL BD Perm/Wash buffer (BD Biosciences, USA) and incubated at 4 °C for 10 min in the dark with 5 μL of Fc Receptor Blocking Solution (Human TruStain FcX, Sony Biotechnology, USA). Next, monoclonal antibodies BV 650-anti-EpCAM (clone 9C4, IgG2b, Sony Biotechnology, USA) were added and incubated at 4 °C for 20 min. The appropriate isotype control antibodies at the same concentration were added to the control sample. After incubation, samples were washed in 1mL Cell Wash buffer (BD Biosciences, USA) at 800× g for 6 min. Then, samples were diluted in 100 μL Stain buffer (Sony Biotechnology, USA). Compensation beads (VersaComp Antibody Capture Bead kit, Beckman Coulter, USA) were used for compensation control. The immunofluorescence was analyzed on the Novocyte 3000 (ACEA Biosciences, San Diego, CA, USA).
The gating strategy was as follows: using forward (FSC) and side scatter (SSC), debris were descriminated, and doublets were also discriminated by plotting FSC area vs. FSC height. Further analysis included only CD45-negative and EpCAM-positive cells. Then, we characterized CTCs by the expression of integrins β3, β4, and αVβ5.
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