Bh 2 ccd microscope

For AlamarBlue (resazurin) proliferation assays, cells were seeded in full medium in 96- well plates. Quantification of initial time 0 was performed the following day, adding 200 μM of AlamarBlue. Cells were then treated according to the experiment and proliferation was measured again after 96 h. In order to perform the colony forming assays, cells were seeded in full medium in 12-well plates and treated the following day according to the experiment. After 10 days, cells were fixed in 4% PFA and stained with Crystal Violet 0.5%. For both the AlamarBlue and colony forming assays, data were analyzed as previously reported [30 (link)]. For the BrdU assay, cells were seeded in full medium on coverslips in a 12-well plate and, after 2 days, treated for 24/48 h according to the experiment set-up. At the end of the treatment, cells were incubated with 1 μg/ml BrdU for 20′ or 30′. Cells were then fixed in 4% PFA and later incubated in 2 N HCl at 37 °C for 15′, causing DNA hydrolysis. After treating with 0.2% Triton and blocking with 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, USA), 3 μg/ml anti-BrdU primary antibody was added O/N. The next day, cells were incubated with secondary antibody and DAPI (Sigma-Aldrich, St. Louis, USA). Images were taken using the Olympus BH-2 CCD microscope. Positive cells were counted using the ImageJ software.

Gelatin degradation assay was performed by coating glass coverslips with Oregon green-488 conjugated gelatin from pig skin (Invitrogen™, Waltham, USA) diluted to a final concentration of 0.2 mg/mL in PBS + sucrose 2%. After coating solidification, gelatin was treated for 15′ on ice with cold glutaraldehyde 0.5% in PBS and then with NaBH₄ 5 mg/mL in PBS for 3′ at room temperature. Coated coverslips were stored at + 4 °C in PBS + P/S 1:50 protected from light until seeding day. 50 k SW48 cells were then seeded and incubated for 24 h to allow cells to settle. For ALKAL1/2 treatment, cells were incubated for 6 h with conditioned medium from HEK293 transfection with ALKAL1/2 plasmids. The coverslips were then fixed with PFA 4% for 15′, blocked for 30′ with BSA3% + 0.1 Triton X-100 and finally stained with Phalloidin-TRITC (Sigma-Aldrich, St. Louis, USA) and DAPI diluted in BSA 0.3% + 0.1 Triton X-100 for 30′. Imaging was performed using the Olympus BH-2 CCD microscope. Images were analyzed using ImageJ software and degradation was measured in the green channel in terms of area of degraded gelatin (without green fluorescence) normalized on nuclei number (DAPI).

For TUNEL assay, cells were seeded in full medium and treated for 48 h according to the experiment. After fixation in 4% PFA, cells were permeabilized with 0.2% Triton and treated for 1 h with the TUNEL reaction mixture (In Situ Cell Death Detection Kit, POD, Enzo Biochem, New York, USA), except for the negative control where the enzyme was not added. Lastly, cells were incubated with DAPI and the coverslip was mounted onto the slide. Images were taken using the Olympus BH-2 CCD microscope, detecting fluorescence in the range of green. Positive cells were counted using the ImageJ software. For the Annexin V/Propidium Iodide (PI) detection assay, 1 × 10⁶ untreated and CZB-treated cells were collected and stained with APC Annexin V and PI (BioLegend, San Diego, USA) for 15′. Detection was performed with Cytoflex S Flow Cytometer and data analysis was carried out with the CytExpert software.