Dulbecco s pbs

HEK293FT cells were seeded in 12-wells plates at 100,000 cells per well and were allowed to adhere overnight. Cells were transfected with 500 ng pcDNA3.1-ARHGEF39 or pcDNA3.1-empty. Cells were counted 48 h after transfection. Total culture medium was removed, centrifuged at 200xg for 3 min and resuspended in 100 ul of Dulbecco’s PBS (Sigma) to count the number of floating cells. Attached cells were detached from the plate with 0.25% trypsin-EDTA (Invitrogen) and resuspended in 1 ml of Dulbecco’s PBS after centrifugation (Sigma). Cells were stained with 0.4% Trypan Blue (BioRad) and counted with the TC20 automated cell counter (BioRad) according to the manufacturer’s instructions. Significant differences in total cell counts and viability percentages were calculated with a two-sided t-test. Viability percentages were arcsine transformed before statistical testing.

Samples were fixed by bringing the cell solution to 4% (vol/vol) paraformaldehyde (Thermo Fisher Scientific) in Dulbecco's PBS (Sigma-Aldrich). Samples were blocked in 2% BSA and 5% goat serum (Sigma-Aldrich) in PBS. Rabbit Histone H3 (citrulline R2+R8+R17) antibody (ab5103; Abcam) was used to immunostain for citrillinated histones. The primary antibody was incubated overnight at 4°C used at a dilution of 1/500 in PBS supplemented with 2% BSA and 5% goat serum. Anti–rabbit Alexa Fluor 568 (A11011; Invitrogen) was used as a secondary antibody. It was used at a dilution of 1/500 and incubated for 1 h at room temperature. Fluorescence microscopy images were taken using a Leica TCS SPE confocal microscope on a 63×/1.40–0.60 oil-immersion objective using Leica Application Suite Advanced Fluorescence v2.7 software (Leica Microsystems). Cells that had extruded DNA were marked yellow as a result of dual staining with both the Sytox green and the anticitrullinated histone antibody.

T. cruzi epimastigotes (TcWT, EcMutT and TcMTH) were treated with 200μM of H₂O₂ for 30 minutes. After incubation, cells were centrifuged at 800 g for 10 min at 25°C and washed twice in DPBS (Dulbecco’s PBS, pH 7.3; Sigma-Aldrich). Parasites (1 × 10⁹ cells/mL) were incubated for 30 min at 28°C in DPBS containing 50 μM DHR (Molecular Probes, Life Technologies, Eugene, OR, USA). After incubation, cells were centrifuged at 800 g for 10 min at 25°C and washed twice in DPBS in order to eliminate non-incorporated DHR. Detection of intracellular Rhodamine 123 (RH 123), the oxidation product of DHR, was performed after exposure to the 0.1mM peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1, Sigma-Aldrich). The detection of intracellular RH 123 was performed using a FACS-Calibur flow cytometer (Becton-Dickinson, Rutherford, NJ, USA).

The secondary plant metabolites (–)-EGCG of Camellia sinensis (“Tea catechin”) was purchased from Cayman Chemical Co., Chicago, IL, USA. α-GH was kindly supplied by Hayashibara Co. Ltd. (Okayama, Japan). Stock concentrations of GH were prepared in Dulbecco’s PBS modified without calcium and without magnesium (Sigma-Aldrich, Saint Louis, MO, USA), and used immediately. For EGCG, 1 ml of the DPBS was added to manufacturer-supplied screw capped glass vials containing 50 mg of the catechin, inverted numerous times to affect placement of the metabolite into solution, used immediately, or placed for 2–3 days at refrigeration temperature (4°C) until use (Zhou et al., 2003 ). Dilution of the GH and EGCG to working strength concentrations coupled with the presence of antibiotics/antimycotic supplements in the cell culture MM, precluded any subsequent bacterial/fungal contamination.

At 7 days post-ischaemia (or at 10 days for the treatment duration study), mice were culled by anaesthetic overdose (Euthatal, 200 mg/ml i.p.; Merial). Approximately 500 μl of blood was collected from the descending aorta and transferred into EDTA-coated tubes. Mice were then perfused transcardially with 20 ml cold phosphate-buffered saline (PBS). The brain was removed from the skull, frozen in isopentane (- 42 °C), and stored at – 20 °C. Brain sections (20 μm) were cut at 500 μm intervals on a cryostat and stored at – 20 °C. Cervical lymph nodes, inguinal lymph nodes, and spleen tissue were harvested and stored in PBS until further manipulation. Spleen and lymph node tissues were mechanically dissociated using the back of a plunger of a 3 ml syringe in approximately 3 ml PBS in a sterile 6-well plate. The resulting cell suspensions were passed through a 70-μm cell strainer and collected in a 50-mL conical tube. Wells and strainers were washed twice with 1X Dulbecco’s PBS (Sigma-Aldrich, #D8537). Spleen and blood samples were re-suspended in 5 mL of 1× Red Blood Cell Lysis Buffer and incubated for 5 min at room temperature (eBioscience, #430054). The lysis reaction was stopped by adding 20 mL of 1X PBS. All samples were then washed twice with 1× PBS, re-suspended in an appropriate volume of PBS and counted using trypan blue to determine total cell concentration and viability.