Universal microplate spectrophotometer

The analysis involved 167 samples of tumor tissue and surgical margin tissue.
The fragments of tumor tissue and surgical margin tissue were weighted and homogenized using a PRO 200 homogenizer (PRO Scientific Inc., Oxford, CT, USA) and sonicated with an ultrasonic cell disrupter (UP 100, Hilscher, Germany). The total protein level was determined using a Universal Microplate Spectrophotometer (µQUANT, Biotek Inc., Winooski, VT, USA).
HHLA2 level was determined by the human HHLA2 ELISA kit (EIAAB SCIENCE INC, WUHAN) with a sensitivity of 0.14 ng/mL. After reaching room temperature by all the reagents, we prepared all the substances, working standards, and samples. The first incubation lasted 2 h at 37 °C after adding 100 uL of standards, blanks, and samples per well. The second incubation lasted 1 h at 37 °C after adding the first detection reagent. After washing processes, we added 100 uL of the second detection reagent, then incubated for 1 h at 37 °C. After washing procedure, 90 uL substrate solution was added and the last incubation lasted 20 min at 37 °C. The last process needed addition of 50 uL stop solution. The optical density of each well was determined using Universal Microplate Spectrophotometer (µQUANT, Biotek Inc., Winooski, VT, USA). (450 nm). The results were recalculated to the corresponding total protein level and presented as ng/mL of protein.

About 1 × 10⁴ transfected PTC cells were seeded in 96-well plates. Cell viability was evaluated with Cell Counting Kit-8 (CCK-8, Dojindo, Japan) at different time points (24, 48, and 72 h) after seeding. After treated with CCK-8 at 37°C for 1 h, PTC cells were used to measure the absorbency at 450 nm using Universal Microplate Spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT, U.S.A.).