Osteosarcoma cell-line SJSA-1 cells (ATCC, Cat# CRL-2098) were cultured in a T75 cell-culture flask with RPMI 1640 medium containing 10% Fetal bovine serum (FBS, Gibco, ThermoFisher, Waltham, MA, USA), and 1% penicillin-streptomycin (Gibco, Thermo Fisher, USA). Cells were seeded at a subculture ratio of 1:10 and regularly passed about once a week (approximately 80% confluent). Culture medium was exchanged every third day and cells were used within 20 passes.
For characterizing low number (<50) cell counting inside a 10 μL pipette tip (Neptune, Cat# BT10), Countess Cell Counting Chamber Slides (ThermoFisher, Cat# C10228) and Nikon TS-100 phase-contrast microscopewere used. For high cell number calculations, cell density was measured by a Countess (ThermoFisher, MA, USA) automated cell counter.
For immunofluorescent imaging of captured tumor cells, CellTracker-Red (ThermoFisher, Cat# C34552) was used to stain the cytoplasm of SJSA-1 cells before the capture, according to the vendor’s instruction. After the capture, the cells were fixed on the Parylene-C membrane inside the capture device by 4% paraformaldehyde with 10 mL PBS wash, then stained by 300 nM DAPI (life Technologies Corporation, Eugene, OR, USA) solution with 20 mL PBS wash. The device was then disassembled, and the membrane was transferred to a glass slide to be mounted under a cover glass using CoverGrip Sealant (BIOTIUM, Fremont, CA, USA, Cat#23005). Finally, the sample was imaged using a Zeiss AXIO Imager M2 Epifluorescent Microscope (Carl Zeiss Microscopy, LLC, White Plains, NY, USA) in the Biomedical Imaging Core at the University of Arizona College of Medicine-Phoenix. The collected images were then analyzed using Zen 2.6 lite software to count the captured cells.
For characterizing low number (<50) cell counting inside a 10 μL pipette tip (Neptune, Cat# BT10), Countess Cell Counting Chamber Slides (ThermoFisher, Cat# C10228) and Nikon TS-100 phase-contrast microscopewere used. For high cell number calculations, cell density was measured by a Countess (ThermoFisher, MA, USA) automated cell counter.
For immunofluorescent imaging of captured tumor cells, CellTracker-Red (ThermoFisher, Cat# C34552) was used to stain the cytoplasm of SJSA-1 cells before the capture, according to the vendor’s instruction. After the capture, the cells were fixed on the Parylene-C membrane inside the capture device by 4% paraformaldehyde with 10 mL PBS wash, then stained by 300 nM DAPI (life Technologies Corporation, Eugene, OR, USA) solution with 20 mL PBS wash. The device was then disassembled, and the membrane was transferred to a glass slide to be mounted under a cover glass using CoverGrip Sealant (BIOTIUM, Fremont, CA, USA, Cat#23005). Finally, the sample was imaged using a Zeiss AXIO Imager M2 Epifluorescent Microscope (Carl Zeiss Microscopy, LLC, White Plains, NY, USA) in the Biomedical Imaging Core at the University of Arizona College of Medicine-Phoenix. The collected images were then analyzed using Zen 2.6 lite software to count the captured cells.
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