Annexin 5 fluos

Isolated neutrophils (5×10⁶/mL) were cultured in 24 or 96-well plates in Iscove’s Modified Dulbecco’s Medium (IMDM) (in some experiments Roswell Park Memorial Medium (RPMI) was substituted) supplemented with 5% autologous serum (in some experiments 10% fetal calf serum (FCS) or no serum was substituted), 1% penicillin and streptomycin, and 1% L-glutamine alone or in the presence of AT7519 (Astex Pharmaceuticals, Cambridge, UK), granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems, Abingdon, UK) or E. coli LPS (Sigma, Dorset, UK) for 24 hours at 37°C, 5% CO₂. At stated time points, neutrophils were resuspended (1:5) in DPBS^-/- supplemented with 25 mM calcium chloride and labelled with Annexin V-FLUOS (Sigma) at 1:500 and 1 µg/mL propidium iodide (PI) before analysis on a BD FACS Scan, FACS Calibur or BD Accuri cell analyzer as described.16 (link) Cytocentrifuge preparations were stained with Diff-Quick (Gamidor, Didcot, UK) to assess for morphological changes of apoptosis.

SW480 cells were plated at a seeding density of 10⁴ × 25 cells/cm² in a T-25 flask. Then cells were transfected with scramble, miR34c-5p mimics, and the vehicle in PBS mock for 48 h. After transfection, cells were double-washed with PBS, detached using trypsin, and centrifuged at 260 RCF for 5 minutes. Next, 0.5 μL of Annexin-V-FLUOS labeled reagent solution, 1 mL of incubation buffer, and 0.5 μL of propidium iodide solution (PI) (Sigma Aldrich, North America) were added to the target groups and incubated for 30 minutes at room temperature in a dark condition. Finally, 700 μL buffer was added to the samples, and the apoptosis rate was determined using flow cytometry (FACSCalibur). The obtained results were then analyzed via FlowJo 7.6.1 (13 (link), 14 )

Collected embryos (1 dpc) of both groups were cultured up to the morula stage as indicated in section Embryo Culture and TNFα Treatment. Randomly selected morulae from both groups of embryos (n = 10) were stained with annexin V and propidium iodide (Annexin-V-FLUOS Staining Kit, Roche Diagnostics, Poland) to confirm the expression of phosphatidylserine as an early sign of apoptosis induction in blastomeres in embryos (28 (link), 30 (link)). Briefly, the embryos were transferred to freshly prepared and preheated microdrops of PBS supplemented with Ca²⁺ Mg²⁺, supplemented with bovine serum albumin (BSA; Sigma, Poland). The embryos were washed three times, each time in a fresh microdrop of PBS (100 μL). Afterward, they were stained at room temperature for 10 min with the Annexin-V-FLUOS (Sigma, Poland) solution reagent with the addition of propidium iodide (20 μL of working-strength solution). After that, the embryos were washed in 10 μL microdrops of fresh PBS with Ca²⁺ Mg²⁺. Before the evaluation under a fluorescence microscope (Leica), the embryos were transferred into microdrops of fresh PBS with the addition of an anti-fade solution (Sigma, Poland). The observations were performed immediately after completed staining.