Facscalibur instrument

Distribution of cells in SubG1, S, and G2/M cell cycle phases was measured based on propidium iodide (PI) staining using standard flow cytometry method. Cell lines were seeded at an initial concentration of 1 × 10⁶ cells mL⁻¹ and incubated for 24 h with or without 30 mM 2-deoxy-D-ribose. Exponentially growing cells were used in a single cell cycle measurement performed in triplicate with fluorescence-activated cell sorting (FACS). After centrifugation (400×g, 4 °C, 5 min), cells were washed with PBS, suspended in 70 % cold ethanol for 1 h, washed again and incubated for 1 h in 0.1 % sodium citrate in PBS containing RNase (10 μg/mL) and 50 μg/mL PI (Sigma-Aldrich). Measurements were performed using FACSCalibur instrument (Becton Dickinson, Franklin Lakes, NJ, USA). Prior to analysis, cells were equilibrated to room temperature. Exactly 10,000 events from each sample were collected in a single cell gate. Aggregates and debris were excluded from analysis by creating the gate on the FL2-W (transit time) versus FL2-A (total cell fluorescence) cytogram according to the standard procedure [77 ]. The percentages of cells in SubG1 phase were determined based on the frequency distribution of DNA content with ModFit LT software (version 3.1, Verity Software House, Topsham, Maine).

The cells were resuspended up to 10⁶ nucleated cells and divided into tubes A and B. In tube A, the cells were incubated with 5 μl CD4-FITC (317407, Biolegend, U.S.A.) and CD185-PE (CXCR5) (356903, BioLegend, U.S.A.) in the dark at RT for 30 min. Then, the tissues were washed twice with PBS for intercellular staining.
Cells in tubes A and B were incubated in 1 ml cold, freshly prepared fixation/permeabilization solution (130-093-142, MACS, GER) in the dark at 4°C for 30 min. After washing twice with permeabilization buffer, the cells were separately stained with Foxp3- Alexa Flour (320013, BioLegend, U.S.A.) and 10 μl Anti-human Aire-APC (130-093-142, MACS, GER) and next incubated for 30 min at RT. The stained cells were detected by flow cytometry immediately.
To guarantee the accuracy of the result, isotype controls were used to determine the gating parameters. FACS Calibur instrument (Becton Dickinson, U.S.A.) was used to conduct flow cytometry.

Apoptotic or necrotic cell death was quantified by flow cytometry using the fluorescein isothiocyanate (FITC)-labelled annexin V and propidium iodide (PI) (Annexin-V-FLUOS Staining Kit [Roche, Germany]) according to the manufacturer’s recommendations. Cell nuclei were stained with Hoechst dye. Cells were harvested after 24 and 48 h incubation with SCS and tamoxifen by using 0.02% trypsin-EDTA, pelleted by centrifugation at 1,000 rpm and washed in phosphate buffered saline. A minimum of 10,000 events were collected and analysed using the FACS Calibur instrument and CellQuest Pro software (Becton Dickinson, USA).

Mouse lung ECs were isolated from three adult mice per genotype, as previously described 13. The purity of ECs prepared with our method has been established 2. Anti‐ICAM2 clone IC2/4 (3C4), (Abd Serotec, Martinsried, Germany); anti‐VECAD clone 11D4.1; (BD Biosciences San Joe, CA, USA) were used to assess EC purity by flow cytometry on a Becton Dickinson, Oxford, UK, FACSCalibur instrument. As a negative control, IgG‐matched isotypes were used (data not shown). Cells were immortalized by two rounds of infection with polyoma virus middle T 14. To ensure maximal Cre‐induced recombination, lung ECs isolated from Cre‐positive mice were transfected with a Cre‐encoding plasmid (pCAG‐Cre‐IRES2‐GFP; Addgene, Cambridge, MA, USA 26646), and cells isolated from Cre‐negative mice were transfected with the corresponding plasmid backbone (pCAG‐GFP; Addgene 11150). Alternatively, immortalized cells were maintained in medium containing 500 nm 4‐hydroxytamoxifen from day 4 after isolation.

Human DLBCL cell lines OCI-Ly01, VAL, OCI-Ly10, SUDHL-8, Farage, and SUDHL-4 cells were purchased from ATCC (Shanghai, China) and cultured in RPMI-1640 medium (Hyclone, Utah, USA) supplemented with 10% fetal bovine serum (FBS) (Ausgenex, Australia), 100 U/mL penicillin, 100 U/mL streptomycin, and 2 mM L-glutamine. Human embryonic kidney 293T cells were maintained in Dulbecco modified Eagle media (DMEM; Hyclone) with 10% FBS. Cells were cultured at 37°C in a 5% CO₂ humidified incubator.
Lentiviral plasmids containing optimized CD300A short hairpin (shRNA) or scramble sequences were produced using 293T cells, and were used to infect DLBCL cells as described [4 (link), 33 (link)]. The sequences for CD300A shRNAs used in the present study included shRNA-1 (5′-GATGTTTCAGAAATGGATCAA-3′), shRNA-2 (5′-CCCAGGGAAGAACTTCACTAT-3′), and a scramble shRNA (5′-CCTAAGGTTAAGTCGCCCTCG-3′).
The surface expression of CD300A in lymphoma cells was determined using a phycoerythrin (PE)-conjugated antibody against human CD300A (clone E59.126, Beckman Coulter, Marseille, France). After staining for 30 min on ice, cells were resuspended in phosphate-buffered saline (PBS) and analyzed using a FACS Calibur instrument (Becton Dickinson, CA, USA). The isotypic antibody (clone 679.1Mc7, Beckman Coulter) was used as control.

Apoptosis was evaluated based on DNA fragmentation using the APO-Direct Assay Staining kit (BD Biosciences, San Diego, CA, USA) as previously described [22 (link)]. In this assay, DNA breaks are labeled with fluorescein isothiocyanate (FITC)-2′-deoxyuridine-5′-triphosphate and cells are analyzed by flow cytometry. MIAPaCa-2 cells were treated with MGDG (25 μM) alone for 24 h, radiation (5 Gy) alone for 12 h, or with a combination of both. The cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS), and then fixed in 1% paraformaldehyde for 15min followed by 70% ethanol overnight at −20 °C. A DNA labeling solution containing FITC was added for 30min; the cells were the resuspended in PBS, and apoptosis was detected by flow cytometry on a FACS Calibur instrument (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed with CellQuest software (Becton Dickinson), and apoptotic cells were quantified as a percentage of the total number of cells.

PAM or vehicle‐treated PRP was stimulated with collagen (4 μg ml⁻¹) or TRAP‐6 (25 μm) in the presence of PGI₂, DEA/NONOate or vehicle. After 4 min, the reaction was stopped with methanol‐free formaldehyde (2% final, Fisher Scientific). Platelets were permeabilized (0.2% Triton X‐100, Sigma) and incubated with anti‐vasodilator stimulated phosphoprotein (VASP)‐P(Ser²³⁹) primary antibody (Enzo Life‐sciences, Exeter, UK), Alexa647‐conjugated secondary antibody (Invitrogen, Paisley, UK) and FITC‐conjugated anti‐CD42b (eBioscience, Hatfield, UK), for 30 min each, in turn, before the platelet pellet was resuspended in 0.9% saline. VASP‐P(Ser²³⁹) immunoreactivity was measured by flow cytometry, using a FACS‐Calibur instrument (Becton Dickinson). Representative histograms are shown in Supplementary Figure 5E,F.