Rhod 2 am

Chlorfenapyr (purity of 97%) was provided by MOLBASE Shanghai Biotechnology Co., Ltd. (Shanghai, China). CHL stock solution was dissolved in DMSO and stored at 4 °C. The stock solution was diluted with the culture medium to the corresponding concentration during the test. Penicillin and streptomycin were obtained from Gibco (Grand Island, NY, USA). Phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), rhodamine 123 (Rh 123), Dulbecco’s modified eagle medium (DMEM), and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO, USA). The Caspase-3/9 assay kit, ROS detection kit, Annexin V/PI apoptosis detection kit, and JC-1 assay kit were obtained from Keygen Biotech Co., Ltd. (Nanjing, China). The superoxidedismutase (SOD) and catalase (CAT) assay kits were all obtained from Beyotime Biotechnology (Shanghai, China). The antibodies LC-3, Bcclin-1, p62, cytochrome-c, Bcl-2, Bax, PARP, OGG1, γH2AX, beta-actin, and the secondary antibody were obtained from Servicebio Co., Ltd. (Wuhan, China). The cell mitochondria isolation kit, Monodansylcadaverine (MDC), Fluo-3 AM, Rhod-2 AM, Lyso-tracker, and Mito-tracker were obtained from Beyotime Biotechnology (Shanghai, China).

Sodium alginate (Alg), sodium polyphosphate (TPP) and calcium chloride (CaCl₂) were got from Beijing Chemical Reagent, China. Glucose oxidase (GO) was obtained from Sigma-Aldrich, USA. Curcumin (Cur) was purchased from Macklin, China. Obatoclax was purchased from Beyotime, China. IR780 was obtained from Alfa Aesar, Ward Hill, USA. Cell counting kit-8 was purchased from Dojindo, Japan. Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin were purchased from Gibco, Thermo Fisher Scientific, USA. Fetal bovine serum was obtained from Wisent, Canada. Lysosome-green and Hoechst33342 were obtained from Dojindo, Japan. Fluorescent probes including Mito-green, Fluo-3AM Ca²⁺ and Rhod-2AM were purchased from Beyotime, China.

The resting RGD⁺ and RGD^- hemocytes were washed twice with modified Leibovitz L15 medium, and the concentration was adjusted to 1 × 10⁶ cells/ml. The specific fluorescent probe DHE or Rhod-2 AM (Beyotime, Shanghai, China) was diluted to 1 μM, mixed with the resting RGD⁺ and RGD^- hemocyte suspensions, respectively, and incubated for 45 min at room temperature in a rotary mixer according to the instructions for the detection of intracellular ROS or Ca²⁺. The stained resting RGD⁺ and RGD^- hemocytes were collected at 800 g and 4°C for 10 min by centrifugation and washed three times with modified Leibovitz L15 medium. The fluorescence signal of the probes was detected by flow cytometry, and the mean fluorescence intensity (MFI) value was used to reflect the intracellular ROS (or Ca²⁺) levels in different populations of hemocytes. There were three replicates for each sample.