Protein tyrosine phosphatase deficiency…
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after 2-hour incubation with the reaction mixture. Protein concentration of cell
lysates was determined and the results are presented as caspase-3
activity/micrograms of total protein.
2.10. Cell viability and cytotoxicity.
Cell viability/damage was determined by two alternative methods: gross
detection of cell viability by using the crystal violet assay (30) and cytotoxicity
assay by measuring lactate dehydrogenase (LDH) leakage into the extracellular
medium (31). For the crystal violet assay, cells were seeded at low density (10
4
cells per well) in 24-well plates, grown for 12 hours with the different treatments
and incubated with crystal violet (0.2% in ethanol) for 20 min. Plates were rinsed
with distilled water and allowed to dry and 1% SDS was then added. The
absorbance of each well was measured using a microplate reader at 570 nm (Bio-
Tek, Winooski, VT, USA). Cytotoxicity was evaluated by the LDH method collecting
both the culture medium and cells that were scraped in phosphate-buffered saline
(PBS) after the different treatments. Then, cells were sonicated to ensure breaking
down the cell membrane to release the total amount of LDH followed by
centrifugation (1.000 ×g 15 min) to clear up the cell sample. 11 microliters of
extract were placed into a well of a 96-multiwell system for the assay. In the same
manner, 11 microliters of each culture medium sample were also deposited in each
96-multiwell. The LDH leakage was estimated from the ratio between the LDH
activity in the culture medium and that of the whole cell content. Fluorescence was
measured at an emission wavelength of 460 nm and an excitation wavelength of
340 nm.
2.11. Quantification of apoptotic cells by flow cytometry.
After APAP stimulation, adherent and non-adherent cells were collected by
centrifugation, washed with PBS and fixed with cold ethanol. The cells were then
washed, resuspended in PBS, and incubated with RNAse A (25 µg/10
6
cells) for 30
min at 37ºC. After addition of 0.05% propidium iodide, cells were analyzed by flow
cytometry.
2.12. Analysis of alanine amino transaminase (ALT) activity.
Blood was collected in tubes containing heparin and diluted 1/30 with
saline (0.9% NaCl). ALT activity was determined by direct measurement with the
Reflotron test (Ref. 10745120, Roche Diagnostics).
2.13. Protein determination.
Protein determination was performed by the Bradford dye method, using
the Bio-Rad reagent and BSA as the standard.
