Reactive oxygen species assay kit

Sodium deoxycholate, propylthiouracil and vitellus powder were procured from Sangon Guangzhou Surui Biotechnology., Ltd. (Guangzhou, China), and cholesterol was obtained from Shanghai Lanji Technology Development Co., Ltd. (Shanghai, China). Total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), superoxide dismutase (SOD), malondialdehyde (MDA) and reactive oxygen species (ROS) assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
HUVECs were a kind gift from Professor HL Sun. Ox-LDL was obtained from Sigma (St. Louis, MO). Dimethylsulphoxide (DMSO) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Aladdin (Shanghai, China). Fenofibrate (FEN; 200 mg/tablet) was procured from Laboratories Fournier (Dijon, France). All other chemicals used were of analytical grade.

Inosine from Corynebacterium spp. was obtained from Sangon Biotech Co., Ltd., (Shanghai, China), and its chemical structure is shown in Figure 1A. LPS was obtained from Sigma-Aldrich (Saint Louis, MO, USA). The assay kits for TNF-α, IL-1β, and IL-6 were obtained from R&D Systems (Shanghai, China). Malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO), lactate dehydrogenase (LDH), glutathione peroxidase (GSH-PX), and reactive oxygen species (ROS) assay kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China).

Mueller‐Hinton agar (MH), MH broth, Lysogeny Broth (LB) broth and DNA rapid extraction kit were purchased from Sangon Biotech (Shanghai, China) Co., Ltd., (NH₄)₂SO₄ was purchased from Beijing Dingguo ChangSheng Biotechnology Co., Ltd (Beijing, China). The cutoff membrane, cell counting kit‐8 assay (CCK8 assay), proteinase K, catalase, trypsin and papain were purchased from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). ATP assay and reactive oxygen species (ROS) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Further, 2‐nitrophenyl β‐d‐galactopyranoside (ONPG) was purchased from Shanghai Macklin Biochemical Co., Ltd., and N‐phenyl‐1‐naphthylamine (NPN) was purchased from Sigma–Aldrich (Shanghai, China).

Intracellular reactive oxygen species (ROS) of the NP cells was measured using a reactive oxygen species assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. Briefly, after washing with PBS for three times and incubation with DCFH-DA (10 μM, diluted by FBS) for 40 min, NP cells were washed with FBS again to remove the uncombined DCFH-DA solution. Thereafter, NP cells were collected by digestion with 0.25% trypsin and 10⁵ cells in each group were used to measure the intracellular ROS that was expressed as fluorescence intensity at an excitation/emission wavelength of 490/585 nm.

ROS in the liver was assessed using the DCFH-DA method and a reactive oxygen species assay kit (Jiancheng Bioengineering Institute, Nanjing, China). Liver tissues were cut up for preparing liver cell suspension. Cell suspensions were centrifuged and resuspended in phosphate buffer saline (PBS). Cells were incubated with DCFH-DA, a fluorescent probe for ROS, for 30 min at 37°C. The fluorescence intensity of ROS was detected by a fluorescence microplate reader (Synergy H1; BioTek, Winooski, VT, USA). The excitation wavelength was 490 nm, and the emission wavelength was 530 nm.

The total intracellular ROS content was analyzed using a reactive oxygen species assay kit (Nanjing Jiancheng Bioengineering Institute, China). After being cultured for 5 days, AF cells were incubated with the DCFH-DA probe (10 μM) for 20 minutes at 37°C in an incubator. Then, AF cells were further washed with the DMEM/F12 medium to remove the unbounded DCFH-DA probe. Finally, relative fluorescence units (RFU, excitation/emission wavelength: 490/585 nm) were detected to reflect the ROS content of AF cells.

To study the effect of RNAi on ROS generation in hemocytes, newly molted 4th instar larvae were fed the NPV-contaminated diet (10 μL of NPV at the concentration of 1.0 × 10⁶ PIB/mL per larva) as the method of NPV challenge. After NPV infection 48 h, the infected larvae were injected with the dsRNA of EGFP, HaTrx2, or HaTrxR1, respectively. After dsRNA injection 48 h, hemolymph of each groups were collected from H. armigera and centrifuged immediately at 4000 × g at 4 °C for 10 min to isolate hemocytes. Then, ROS production was measured with Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Hemocytes were incubated with DCFH-DA (2, 7-dichlorofuorescin diacetate) at a final concentration of 10 μM for 20 min. Hemocyte morphology was observed using a OLYMPUS BX61 (Olympus, Tokyo, Japan) laser scanning confocal microscope. ROS production in hemocytes was measured fluorometrically at excitation and emission wavelengths of 488 and 525 nm, respectively.
Usually, ROS damage caused lipid peroxidation in the organism. As a terminal product of lipid peroxidation, MDA was measured to evaluate the degree of lipid peroxidation in the hemolymph using MDA Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).