Annexin 5 fitc apoptosis detection kit 1

Recombinant strain E. coli BL21(DE3)/pET24a(+)-csnA for expressing the chitosanase was previously constructed in our laboratory [16 (link)]. Human IAPP (> 95%) was synthesized by Gen Script (Nanjing, China). Thioflavin T (ThT) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kit I was obtained from Vazyme Biotech (Najing, China). Chitosan (C90% deacetylated) was purchased from Qingdao MdBio, Inc. (Qingdao, China). The silica gel plates 60F 254 for thin layer chromatography (TLC) was purchased from Merck (Darmstadt, Germany).

For spleen tissues, partial spleen blocks were minced and passed through a 70-μm cell strainer. Dispersed tissues were washed in phosphate-buffered saline 3 times and then directly stained with FITC-conjugated Annexin-V and propidium iodide (PI) for 10 min to analyze apoptosis using the Annexin V-FITC Apoptosis Detection Kit I (Vazyme, A211-01/02) according to the manufacturer’s instructions.
For spleen cells cultivated ex vivo, cells washed in phosphate-buffered saline 3 times were respectively stained with APC-conjugated antibody against CD79a (Abnova, MAB4515) and PE/Cy5-conjugated antibody against CD3 (Abcam, ab25535) at 37 °C for 1 hour. After cells were washed in phosphate-buffered saline 3 times again, the Annexin V-FITC Apoptosis Detection Kit I was used to analyze apoptosis as described above.