Lsr 2 facs machine

The 8–10-week-old NSG mice were injected i.v. with 1 × 10⁶ genetically modified human T cells. On day 28 after T-cell injection, 5 × 10⁵ MOLM-13 cells were given i.v. to the mice and treatment was started 5 days later. For this purpose, 250 ng per g mouse body weight of the TM was injected i.p. twice a day over 2 consecutive days. Mice were killed when severe derogation of health occurred because of tumor development and single-cell suspensions from bone marrow obtained from femur and tibia of the left hind leg were prepared. Erythrocytes were removed by lysis and nucleated cells were stained with anti-mouse CD45.1/PE-Cy7 (clone A20, eBioscience), anti-human CD3/APC-eFluor 780 (clone SK7, eBioscience), CD19/APC (clone HIB19, BD Biosciences), CD33/PE (clone HIM3-4, eBioscience) and CD45/AlexaFluor 700 (clone HI30, BioLegend) mAbs. Doublet discrimination was routinely carried out and dead cells were excluded by 4',6-diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich, Steinheim, Germany). All measurements were performed on a BD LSRII FACS machine (BD Biosciences). Data analysis was realized using FlowJo software (Tree Star Inc., Ashland, OR, USA).

HUVEC were treated with 1 μM CA4P for 60 min +/- 30 min 1 μM S1P pre-treatment. Following treatment cells were washed in PBS then detached using 2 mM EDTA at 37°C for 15-20 minutes. Cell suspensions were collected, washed twice in PBS then resuspended in ice-cold Immunomix containing mouse anti-human CD144 (BD Pharmingen, BD555661) antibody at a dilution of 1:100. Cells were incubated at 4°C for 1 h, then washed twice and resuspended in ice cold Immuonomix containing Alexafluor 555 (Invitrogen) at 1:200. Cells were incubated at 4°C for 30 mins then washed twice in cold PBS and resuspended in 300 ul cold PBS before analysis on an LSRII FACS machine (BD).

AP and SSEA1 staining were examined using Stemgent Alkaline Phosphatase Staining Kit II (Stemgent) and Phycoerythrin conjugated mouse anti-SSEA1 antibodies (R&D Systems), respectively, following the manufacturer’s recommendations. SSEA1 marker expression was evaluated on a LSR II FACS machine (BD Biosciences).

Freshly isolated lymph node-derived lymphocytes from naive Brown Norway rats were collected as responder cells for MLRs. Cryopreserved liver-resident immune cells, isolated from the livers of control and experimental Lewis rats, were recovered to serve as allogeneic stimulator cells. In Phosphate buffered saline (PBS), at a concentration of 1 × 10⁶ cells/ml, stimulators were labeled with a 1:1,000 dilution of Cell Trace Violet (CTV), while responders were separately labeled with a 1:1,000 dilution of Cell Trace Far Red (CTFR) (Thermo Fisher Scientific, Waltham, MA, USA). Stimulator cells then received 20 grays of irradiation prior to culture. In a 96-well plate, 2 × 10⁵ responders were plated with 0.5 × 10⁵ irradiated stimulators in RPMI media supplemented with 10% fetal bovine serum (FBS). Negative controls were established using irradiated CTFR-labeled leukocytes derived from naive Brown Norway livers. Non-specific stimulation using activating plate-bound anti-CD3 (1ug/mL; BD Biosciences) and soluble anti-CD28 (1 µg/ml; BD Biosciences) served as positive controls. All experimental conditions were performed in triplicate. After 4 days of culture at 37°C, cells were collected and flow cytometry data were acquired using an LSR II FACS machine (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, San Carlos, CA, USA).