Atplite kit

Cultured rat fibroblasts, which were established from the subcutaneous tissue under the back skin of a healthy Wistar S/T rat (seven weeks old, 200 g), were retrieved in the form of a single cell suspension with D-MEM medium (Wako Pure Chemical) containing 10% bovine serum. The cell suspension was diluted with the same medium to 1.5 × 10⁴ cells/mL. This suspension was poured at 100 μL/well into the coated wells in the plates described above so that there were 1.0 × 10⁴ human cells/well (n = 4) as a control. The cell suspension was also poured into a humidified incubator with 5% CO₂ at 37°C.
For the cell culture, we used 24-well culture plates with wells 15 mm in diameter without a coating (Becton, Dickinson and Company Ltd., Franklin Lakes, USA). The 24 wells were divided into three groups: the SL group, WL group, and NL group. Fifty mg of sodium alginate and the calcium gluconate and/or physiological saline solutions were combined in the same proportions by weight. The type and volume of solution are summarized in Table 4. One, three, five, and seven days after seeding, the viable cell number in each well was counted with the ATP assay using an ATP Lite Kit (Perkin Elmer, Waltham, USA). For each time point, four wells for each experimental group were examined.

Cells were seeded in 96-well plates in triplicates and cell proliferation was measured with an ATPlite kit (Perkin Elmer, Boston, MA) [41 (link)].

We purchased antibodies against p21 (mouse mAb) and p27 (mouse mAb) from BD Transduction Labs (Gibbstown, NJ), antibodies against DEPTOR, AKT, pS6, 4EBP1, p4EBP1 cleaved Casp3 and pAKT polyclonal antibodies from Cell Signaling Technology (Danvers, MA), S6 (mouse mAb) and NRF2 from Santa Cruz Biotechnology (Santa Cruz, CA), PHLPP1 polyclonal antibody from Bethyl Laboratories (Montgomery, TX) and β-Actin (mouse mAb) from Sigma (St. Louis, MO), Ki67 from BD Biosciences (Gibbstown, NJ), Dab1 and BrdU (rat mAb) from Abcam (Cambridge, MA), FLNA from Abnova (Walnut, CA). DEPTOR siRNA, PHLPP1 siRNA and control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). In Situ Cell Death Detection Kit was purchased from Roche (Indianapolis, IN). ATPlite kit was obtained from Perkin Elmer (Boston, MA).

ATP, the energy fuel of the cell, is also a coenzyme involved in numerous metabolic reactions. ATP content in the head, midgut and semen was determined using ATPlite kit (PerkinElmer) based on luminescence measurements produced by the oxidation of D-luciferin by luciferase that involves 1 molecule of ATP and O₂^{84 (link), 93 (link)}.

Cell viability was measured using an ATPLite Kit (cat# 6016941; PerkinElmer, Waltham, MA). The assay was performed according to the manufacturer's instructions. Luminescence was read using a multi scan plate reader (Fisher, Waltham, MA).

ATP levels in BALF were measured using ATPLite kit (Perkin Elmer), as previously described [4 (link)]. BALF cytokine contents were determined by ELISA (R&D Systems, Minneapolis, USA), as described by the manufacturer. BALF collagen content was measured by Sircol assay (Biocolor, Carrickfergus, U.K.).