Uv2010 spectrophotometer

HT-115 cell (human colon cancer), human mesenchymal stem (hMSCs) and Vero (normal monkey kidney cell) cells have been purchased from American type culture collections (ATCC, USA). Roswell park memorial institute (RPMI-1640) medium, trypsin, EDTA and all other cell culture materials have been purchased from Gibco, Paisley, UK. Fetal bovine serum (FBS) was purchased from Hiclone, Germany. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO, USA). cDNA synthesis kit and SYBR Green PCR master mix were purchased from Qiagen, Hilden, Germany. Deionized water was collected from Direct-QUV3 Multipore water purification system (Millipore, Burlington, MA, USA). Fine chemicals related to molecular biology experiment have been purchased from Sigma-Aldrich chemical company (St. Louis, MO, USA). Spectrophotometric measurements were performed with UV2010 spectrophotometer (Hitachi, Germany).

The larvae were dissected after 24 h and midgut was extracted in 0.2 M sodium phosphate buffer (pH 7.5). The midguts were removed and homogenized in 0.1 M glycine-NaOH buffer, pH 10, containing 1 mM EDTA. The homogenate was centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was used as enzyme for serine protease and trypsin activities. To estimate the serine protease activity of insect midgut, the method followed by Hegedus et al^{13 (link)}
was followed using azocasein as a substrate. The midgut supernatant 0.04 mL was put in a test tube and 0.3 mL of 1% azocasein solution (prepared in 0.05 M glycine-NaOH buffer, pH 10) was added to it. After incubation for 15 min at 28°C, 0.34 mL of 10% TCA was added to it. The reaction mixture was incubated for 1 h at room temperature and centrifuged at 12,000 rpm for 10 min. The supernatant was collected and 0.68 mL of 1 M NaOH was added to it. Absorbance was read at 495 nm on a HITACHI UV-2010 spectrophotometer.
The serine protease activity (SP) was calculated by subtracting the azocasein blank absorbance from sample absorbance, divided by incubation time in min, multiplied by 1000.
$$\text{SP}=\frac{{\text{Abs}}_{\left(\text{sample}\right)}–{\text{Abs}}_{\left(\text{blank}\right)}}{\text{Incubation}\hspace{0.17em}\text{time}\hspace{0.17em}\left(min\right)}\times 1000$$
Units are tryptic activity (mu) per min of incubation per mg protein (mu min⁻¹ mg¹
protein).