Macs tissue storage solution

This study had been approved by the Ethics Committee of Shenzhen Bay Laboratory, Ethics Committee of The Third People’s Hospital of Shenzhen. Participants were recruited from The Third People’s Hospital of Shenzhen and Peking Union Medical College Hospital. Written informed consents were obtained from all participants. For each subject, 10 ml peripheral blood was collected using EDTA-containing tubes, stored at 4 °C and processed within 4 h^{75 (link)}. Briefly, blood was centrifuged at 1600 g, 4 °C for 15 min, then the plasma portion was harvested and re-recentrifuged at 16,000 g, 4 °C for 15 min to remove blood cells. Plasma samples were stored at −80 °C until further usage. Tumor samples (1 from primary colon tumor, 1 from liver metastasis) were collected during surgical resections; the specimens were immediately washed using physiological saline, then stored in MACS Tissue Storage Solution (Miltenyi Biotec, #130-100-008) and implanted into immunocompromised NOD/SCID gamma (NSG) mice^{76 (link)} (8-week old) within 48 h. Animal studies were conducted according to protocols approved by the Institutional Animal Care and Use Committee, Southern University of Science and Technology. Mice were housed under specific pathogen-free conditions with a 12 h light/dark cycle, at a temperature of 20–26 °C, and a relative humidity of 40–70%; mice were fed a standard mouse chow diet.

Decidual tissues were washed with saline, collected in ice-cold MACS Tissue storage solution (catalog no. 130-100-008, Miltenyi, Germany) and dissociated into single cells. CyTOF analyses were performed by PLT Tech Inc. (Hangzhou, China). In brief, cells were stained 5 min with 5 µM 194Pt cisplatin (Fluidigm) for viability in phosphate buffered saline (PBS). After being blocked with PBS containing 5% goat serum and 30% bovine serum albumin (BSA) for 30 min at 4°C, cells were stained with cell-surface antibodies for 30 min at 4°C and washed twice with PBS containing 2.5% BSA. The Foxp3 Fixation and Permeabilization kit (eBioscience) was used according to the instructions of the manufacturer, and cells were incubated overnight in 1.6% paraformaldehyde (PFA) PBS with 100 nM iridium nucleic acid intercalator (Fluidigm) and lastly incubated with intracellular antibodies in permeabilization buffer for 30 min at 4 C after being washed twice with Foxp3 permeabilization buffer and twice with FACS buffer. Cells were washed, resuspended at a concentration of one million cells/ml with water containing EQ Four Element Calibration Beads (Fluidigm), and analyzed within 12 h on a Helios CyTOF System (Fluidigm) at an event rate of <500 events/s.