Lyso id red lysosomal detection kit

A LYSO-ID Red lysosomal detection kit (Enzo Life Sciences, Farmingdale, NY) was used for the lysosome detection in PMNs or macrophages. Lysosomal staining was performed according to the manufacturer’s recommendations. Briefly, PMNs or macrophages collected from DKA mice were suspended in 250 μl of HBSS with 10% mouse heat-inactivated serum at a concentration of 2 × 10⁶ cells/ml. The phagocytes were incubated with or without 250 μl of R. delemar spores and R. delemar spores + either preimmune IgG or anti-CotH3 IgG (3 μg/ml) for 30 min at 37°C with gentle shaking (1 rpm). A LYSO-ID Red detection reagent (0.5 μl) (Enzo Life Sciences) and Hoechst 33342 nuclear stain (0.5 μl) were added to 500 μl of cells. The samples were then incubated for an additional 30 min at 37°C and washed with 100 μl of 1× assay buffer. The stained cells were imaged with a Leica confocal microscope using a Texas Red filter set for lysosomal imaging and a 4′,6-diamidino-2-phenylindole (DAPI) filter set for Hoechst 33342 staining nuclear imaging.

Thymidine, Chlorpromazine, Colchicine and indomethacin were purchased from Solarbio (Beijing, China). Hochest33342, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) and Lactate dehydrogenase (LDH) Release Assay Kit were purchased from Beyotime Biotechnology (Jiangsu, China). Lyso-ID Red Lysosomal Detection Kit and Nucleolar-ID Green Detection Kit (GFP-Certified) were purchased from ENZO Life Science (Missouri, USA). Cell Cycle Detection Kit was purchased from Keygen Biotech (Nanjing, China). TIANpure Midi Plasmid Kit was purchased from Tiangen Biotech (Beijing, China). MCF-7cells and Escherichia coli containing pcDNA3.1-EGFP were provided as gifts from the Department of Pharmacology Teaching and Research Department at China Medical University. CBA was purchased from Alfa Aesar (MA, USA). 2, 2, 4, 6, 7-Pentamethydihydrobenzofuran-5-sulfonyl chloride (Pbf-Cl), CAR, and CHL were purchased from Sinopharm Chemical Regent (Shanghai, China).