Micro mda assay kit

To assess lipid oxidation levels in fetal brains, MDA levels were measured by the Micro MDA Assay Kit (BC0025, Solarbio) according to the manufacturer's protocol. The absorbance of each sample was measured at 450, 532, and 600 nm, and the MDA content could be calculated according to the protocol.

After 3 days of NaCl treatment and 7 days of drought treatment, ten leaves were collected from each line and used to measure MDA content and REL. MDA was detected using the micro MDA assay kit (Solarbio, Bejing, China) according to the manufacturer’s instructions. The fresh weight (W) was determined for leaf samples, followed by homogenization in 1 mL extract buffer in an ice bath and centrifugation at 8000× g for 10 min at 4 °C. The supernatant was used to measure absorbance of A532, A600 and A450 with NanoDrop^TM One^C (Thermo Scientific, Waltham, MA, USA). The MDA content was calculated using the following formula: MDA content (nmol g⁻¹) = 5 × (12.9 × (A532 − A600) − 2.58 × A450) ÷ W.
The REL was measured as described by Yu et al. [57 (link)] with minor modifications. Leaves were sampled, rinsed with deionized water and dried with filter paper. A 0.3 g sample of leaf was placed into a 50 ml tube with 20 ml deionized water for 5 h. The conductivity (C₁) was measured with a conductivity meter model DDS-11 (Biocotek, Ningbo, China). Subsequently, the samples were boiled for 15 minutes, cooled at room temperature and used to measure conductivity (C₂) again. REL was calculated using the following formula: REL = (C₁/C₂) × 100%.

The MDA content from plant samples was measured using Micro MDA Assay Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Each sample was replicated at least 3 times.
A total of 0.1 g of sample was ground with a small amount of quartz sand, calcium carbonate powder, and 2.5 mL of anhydrous ethanol and then transferred into a 10 mL centrifuge tube, and 7.5 mL of anhydrous ethanol was added. After centrifugation at 4 °C and 5000× g for 10 min, 200 μL of the supernatant was applied to determine absorbance at 665 and 645 nm. The chlorophyll content of the sample was calculated according to the simplified formula: chlorophyll content (mg·g⁻¹, FW) = 0.663 A665 + 0.808 A649. Each sample was replicated at least 3 times.

Intracellular MDA levels in the cells were measured using micro MDA assay kit (Solarbio, China) following the instruction by the manufacturer. Briefly, the cells of each group were collected and disrupted by an ultrasonic cell pulverize. The cell suspension was centrifuged and then 100 μL sample was added for the measurement, followed by the addition of 400 μL of the MDA test solution. After mixing and reacting in a 100°C-water bath for 30 min, the mixture was cooled to room temperature and centrifuged. Next the supernatant was taken out and measured absorbance at 450, 532, and 600 nm wavelength with a full-wavelength microplate reader (Thermo, United States).

The seeds of WT, atspt4-2-1, and OE10 were germinated on MS medium without or with 120 mM NaCl for 5 days, and then the whole plants were used for the measurement of proline and malondialdehyde (MDA) contents and antioxidant enzyme activity. Proline content was measured using a Proline Content Determination Kit (CAS: BC0295, Solarbio, Beijing, China). MDA content was measured using a Micro MDA Assay Kit (CAS: BC0025, Solarbio, Beijing, China). Superoxide dismutase (SOD) activity was measured using a SOD Assay Kit (CAS: BC0175, Solarbio, Beijing, China). Peroxidase (POD) activity was measured using a Micro POD Assay Kit (CAS: BC0095, Solarbio, Beijing, China).

The H₂O₂ content of seedlings was measured by previously described methods [30 (link)]. Approx. 0.5 g of samples were cut into small pieces and homogenized in an ice bath with 5 mL 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12, 000 g for 20 min at 4 °C. 0.5 mL of the supernatant was added to 0.5 mL 10 mM potassium phosphate buffer (pH 7.0) and 1 mL 1 M KI. The absorbance of the supernatant was read at 390 nm. The content of H₂O₂ was determined by a standard curve.
Electrolyte leakage (EL) was calculated by measuring the conductivity [31 (link)]. Approx. 0.5 g of samples were cut into small pieces and transferred to 10 mL deionized water. The conductivity (C1) was measured after standing for 2 h at room temperature. After measuring the conductivity, the samples were boiled for 15 min to achieve 100% ion leakage (C2). Relative conductivity = C1/C2 × 100%.
Lipid peroxidation was estimated by measuring the malondialdehyde (MDA), according to the protocol of Micro MDA Assay Kit (Solarbio, Beijing, China).

The upper leaves (3rd to 8th from the top) were collected from the WT P. × canescens and two PeGRP2-overexpressing lines, L-7 and L-8, after 15 days of NaCl treatment (0 or 100 mM). The REL was calculated from the initial relative conductivity (EC1) before boiling and the final conductivity (EC2) after boiling: REL (%) = (EC1/EC2) × 100% [7 (link)]. The MDA content was determined using the micro MDA assay kit (BC0025) (Beijing Solarbio Science & Technology, Beijing, China). Fresh leaves (0.1 g) were ground into a homogenate by adding 1 mL of extraction buffer and then centrifuged at 8000× g for 10 min at 4 °C, and the supernatant was collected. The absorbance values (ΔA) were measured at 532 nm and 600 nm using a microplate reader. The MDA concentration (nmol g⁻¹) was calculated as 32.258 × (ΔA₅₃₂ − ΔA₆₀₀)/0.1.