Proline pro content assay kit

Proline content was measured using Proline (PRO) Content Assay Kit (BC0290, Solarbio, Beijing, China) following the manufacturer’s protocol. Briefly, 10-day-old seedlings from ½ MS agar plates were transferred into ½ MS liquid medium at 12 h before treatments and then treated overnight with 200 mM of NaCl. 100 mg of seedlings were weighted and homogenized with 1 mL of extraction buffer in mortar on ice. Then, the extraction procedure was followed the manufacturer’s protocol. Proline content was determined by reading the optical density of the sample at 520 nm using a spectrometer.

Blood samples were centrifuged at 3000 g for 10 minutes at room temperature to separate serum. ALT, AST and ammonia concentrations were evaluated with a dry chemistry analyser (FUJI DRICHEM 7000 V, FUJIFILM, Tokyo, Japan). Fecal samples (100 mg) were suspended in 1 ml PBS, and supernatants were obtained by centrifuging at 13000 g for 10 minutes. The cerebrum of mice was fully ground in 500ul PBS, and the supernatants were spun at 13000 g for 10 minutes at room temperature to obtain the supernatants. The fecal and brain ammonia levels were also detected by the dry chemistry analyser. The acetate levels were determined with Acetate Colorimetric Assay Kit (Sigma-Aldrich, US). Proline levels were detected using the Proline (PRO) Content Assay Kit (Solarbio, China).

The differences in the physiological responses of the ‘268’ and ‘334’ lines under control and heat stress treatments were measured during CK, HT-4, HT-8, HT-10, and RC-4 periods. In accordance with the manufacturer’s instructions, the corresponding kits (Solarbio Life Sciences, Beijing, China) were selected to determine various physiological indicators. The Catalase (CAT) Activity Assay Kit (Cat#BC0200; Solarbio, Beijing, China), Superoxide Dismutase (SOD) Activity Assay Kit (Cat#BC0170; Solarbio, Beijing, China), Proline (Pro) Content Assay Kit (Cat#BC0290; Solarbio, Beijing, China), Malondialdehyde (MDA) Content Assay Kit (Cat#BC0020; Solarbio, Beijing, China), and Plant Soluble Sugar (SS) Content Assay Kit (Cat#BC0030; Solarbio, Beijing, China) were used for subsequent spectrophotometric measurements. Three biological replicates of each sample (n = 3) were assessed.