Pcs 800 017

Human TNBC cells MDA-MB-231 (HTB-26) and MDA-MB-468 (HTB-132) and CD8⁺ cytotoxic T cells (PCS-800–017) were procured from ATCC (Manassas, VA, USA). Another TNBC cell line, MDA-MB-453 (BNCC340797), was acquired from BeNa Culture Collection (Beijing, China). The TNBC cells were cultured in L-15 medium along with 10% fetal bovine serum (FBS) in a 37℃ incubator, and the CD8⁺ T cells were recovered and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% FBS.

The cytotoxicity in LUAD cells was determined by a lactate dehydrogenase (LDH) kit (C0016; Beyotime). LUAD cells were used as target cells, while the CD8⁺ cytotoxic T cells (PCS‐800‐017; ATCC) were used as the effector cells. In short, 4 × 10⁵ LUAD cells and 6 × 10⁵ T cells were seeded into 96‐well plates. After 24 h of incubation,²⁰ cells were centrifuged at 500g, and 120 μL cell culture supernatant was collected, which was incubated with 60 μL LDH avoiding light exposure for 30 min. The OD₄₉₀ value was read by the microplate reader. The cytotoxicity in cells (%) = (LDH release in mixture of target and effector cells – LDH release in effector cells)/LDH release in target cells × 100.

The skins were stored in formaldehyde 15% for 48 h. Furthermore, alcohol was used as a dehydration agent with concentration of 70%, 80%, and 96%. Xylol was used for clearing process and continued making paraffin block with 60°C of the temperature. The skin tissue that has received paraffin blocks then sliced using a microtome machine and then transferred into a water bath before being placed on a glass object. Immunohistochemistry staining was used primary antibody caspase-3 anti-rat (PE Active Caspase-3 Apoptosis Kit BD Pharmingen^™ with CAS number 550914) and CTL (ATCC^® PCS-800-017^™) for 1 h in 27°C. The dilution was given 10 µl for caspase-3 and 0.1 ml for CTL test. Caspase-3 was executor primary antibody [17 ]. Then, the specimen washed in phosphate buffered saline (PBS) with a pH of 7.4 for 3 times every 5 min. The next preparations were added streptavidin-horseradish peroxidase for 60 min in 27°C and washed in PBS with pH 7.4. Then, the specimens were added chromogen 3,3-Diaminobenzidine tetrahydrochloride for 20 min and washed with aquadest for 5 min.