Cytation 5 cell imaging reader

Schwann cells or fibroblasts were seeded on 8-well chamber slides (Ibidi) coated with poly-L-lysine and laminin, as described.⁹ (link) Cells were harvested 24 to 48 h later and fixed in 4% paraformaldehyde (EM Sciences) for 15 min at RT. Fixed cells were permeabilized in PBS containing 1% bovine serum albumin and 0.2% Triton X-100 for 30 min at RT, and further incubated with anti-p75 NGFR overnight at 4°C in the dark. The next day, cells were washed in PBS containing 1% BSA two times for 5 min at RT, incubated with anti-phalloidin for 1 h in the dark at RT, and washed again as described above. Cells were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo) and coverslipped using Gold Seal Cover Glass (EM Sciences). Images were acquired using a Cytation 5 cell imaging reader (BioTek).

Indicated number of cells were seeded in each well of 96‐well plates for cell viability assay to monitor cell viability at indicated time periods using Cell Counting Kit 8 (CCK8, Bimake, B34304) according to manufacturer's instruction. Briefly, at indicated time points post‐cell seeding, 10 µL CCK8 solution was added into each well and incubated in the culture incubator (37 °C with 5% CO₂) for 3 h. After thorough mixing, absorbance at 450 nm was measured using the BioTek Cytation 5 Cell Imaging reader. Combination index (CI) of co‐treatment of 2‐BP and sorafenib was calculated by CompuSyn software (ComboSyn, Inc).

2000 indicated cells were seeded in each well of 96‐well plates for MTT assays to monitor cell viability at indicated time periods using a method adapted from https://www.thermofisher.com/us/en/home/references/protocols/cell‐culture/mtt‐assay‐protocol/vybrant‐mtt‐cell‐proliferation‐assay‐kit.html. Briefly, at indicated time points post‐cell seeding, 10 µL MTT solution was added into each well and incubated in the culture incubator (37 °C with 5% CO₂) for 4 h. Then, medium was removed and 100 µL dimethyl sulfoxide (DMSO) was added into each well to dissolve the formazan crystal and incubated for 10 min at 37 °C. After thorough mixing, absorbance at 540 nm was measured using the BioTek Cytation 5 Cell Imaging reader.

Following post-imaging, a proliferation assay was conducted via Promega’s Celltiter-Glo^Ⓡ Luminescent Cell Viability Assay (Madison, WI, USA). At 50 µM and 200 µL per well, the end culture 96-well plate was transferred onto a luminesce-compatible well plate and the viability read was captured using the Cytation 5 Cell Imaging Reader (BioTek Instruments, Inc., Winooski, VT, USA). The viability results were then further analyzed using GraphPad Prism 8.3.0 (San Diego, CA, USA).
Similarly, for the ultra-large spheroids, cell viability reagent was prepared according to the CellTiter-Glo^Ⓡ 3D Cell Viability Assay protocol [23 ]. The CellTiter-Glo^Ⓡ 3D Reagent was thawed at 4 °C overnight and equilibrated to room temperature prior to use. Samples were transferred from petri dish to a 96-well plate with clear bottom wells and microtissues prior to viability reagent application; the samples were suspended in culture media. After application of CellTiter-Glo^Ⓡ 3D Reagent, the plate luminescence was measured by a BioTek Synergy/Neo2 (Winooski, VT, USA) plate reader and recorded.

Fibrin polymerization was measured by monitoring turbidity changes with time at 350 nm at 37 °C for 1 h using a UV–Vis spectrophotometer (Cytation 5 Cell Imaging Reader, BioTek). Fibrin polymerization was initiated by the addition of 100 μL of 2 × coating buffer (0.14 M NaCl, 2 mM CaCl_2, and 44 mM Hepes buffer at pH 7.4) containing 0.16 NIH U/mL thrombin into 100 μL of 2 mg/mL oxidized or fibrinogen control solution. PBS was used as the blank. The turbidity changes of the samples in the process of gelation were kinetically measured for 1 h. The turbidity curves were characterized using the following parameters: (1) the lag time, measured as the time elapsed until an increase in absorbance was seen, which reflects the time to the start of lateral fibril aggregation after cleavage of fibrinopeptides by thrombin; (2) the maximal slope (Vmax), calculated as the slope of the steepest part of the polymerization curve, which represents the rate of lateral protofibril association the rate of protofibril aggregation into fibers; (3) the maximal turbidity of the growing clot, recorded 60 min after polymerization was initiated, which reflects fibrin fiber diameter and the number of protofibrils per fiber (turbidity was correlated with the thickness of an individual fiber)^{22 (link)}.