2 3 5 triphenyltetrazolium chloride ttc

All of the animal experiments mentioned in this article received approval from the Guangdong Medical University Ethics Committee for Animals following the NIH Care Guidelines and usage of experimental animals. Male Sprague Dawley (SD) mice (weight range from 280 to 320 g) were anesthetized with an intraperitoneal dose of ketamine (75 mg/kg) and 3% isoflurane. The transient MCAO model experiment was carried out as reported earlier [20 (link)]. After anesthesia, the remaining internal and external carotid arteries were surgically removed via an endoscopic midline incision. After 24 h, the middle cerebral artery was sealed with a round nylon (4-0 suture) tip, and the junction was detached to allow blood flow restoration. The control rats (sham group) underwent identical operations, but the middle cerebral artery was unblocked. The experimental animals that underwent surgery were treated with heating pads to keep their body temperatures at 37 ± 0.5 °C. The rats were sacrificed 24 h after MCAO modeling. Subsequently, infarcted brain tissue was isolated and cut into 3-mm-thick sections for staining with 2,3,5-triphenyl tetrazolium chloride (TTC) (Sigma, MO, USA).

Microbroth dilution on 96-well microplates was used to determine the minimum inhibitory concentration (MIC) of each essential oil following the procedure published by Eloff [23 (link)] with minor modifications. In brief, in each microplate row, decreasing amounts of each essential oil were prepared in DMSO using the serial twofold dilution procedure. The microplates were then incubated at 37°C for 24 hours with 20 μL of bacterial suspensions adjusted to 0.5 McFarland and 160 μL of Mueller–Hinton broth (MHB, Biokar, Beauvais, France). After that, the bacterial growth was examined by incubating for 30 minutes at 37°C with 40 μL of 2, 3, 5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, Switzerland) at a concentration of 0.2 µg/mL. The TTC uses a red dye to stain the bacteria, indicating which wells have bacterial growth. The MIC was determined using microplate wells with the lowest concentration of essential oils and no evident bacterial growth. The minimal bactericide concentration (MBC) test [24 (link)] was determined by subculturing 10 μL from a microplate well that did not show bacterial growth on Mueller–Hinton Agar (Biokar, Beauvais, France), and then incubating the plates at 37°C for 24 hours. The MBC was defined as the lowest concentration that did not cause any growth in the medium. The reference test in this investigation was chloramphenicol (Sigma-Aldrich).

Scu (98% pure) was purchased from CHENGDU MUST BIO-TECHNOL CO., LTD. (Chengdu, China). Scu was dissolved in saline before use. 2,3,5-Triphenyltetrazolium chloride (TTC) was bought from Sigma Chemical Co. (St. Louis, MO). Antibodies against ACE, AT1R, TNF-α, IL-1β, IL-6, and β-actin were obtained from Cell Signaling Technology (1:10000, Beverly, MA, USA). All other chemicals and reagents were of analytical grade.

Potato dextrose agar (PDA) and broth (PDB), Mueller-Hinton agar (MHA) and broth (MHB), Sabouraud dextrose agar (SDA), gentamicin, nystatin, and sodium dodecyl sulfate (SDS) were purchased from HiMedia (Mumbai, India). Other materials were obtained from different manufacturers such as sodium hypochlorite solution (available chlorine 4%) from Fisher Scientific (Mumbai, India); Precoated aluminum-backed TLC silica gel plates (60 F₂₅₄) from Merck (Darmstadt, Germany); HPLC grade solvents like ethyl acetate; hexane and methanol from SDFCL (Mumbai, India); and Silica gel 60–120 mesh from Qualigens (Mumbai, India). The ITS 1 and ITS 4 primers, 2,3,5-triphenyl tetrazolium chloride (TTC), and Thiazolyl Blue Tetrazolium Bromide (MTT) were procured from Sigma-Aldrich (Missouri, USA).

Overall, 3-month-old male Dnm1l^+/- mice and their littermates were submitted to myocardial I/R in vivo. Dpr1^+/- and WT mice were anesthetized by means of intraperitoneal sodium pentobarbital injections (80mg/Kg; Ceva Santé Animale) and received an injection of heparin (100IU/Kg, Heparine Choay^®, Sanofi aventis, Paris, France) prior to thoracotomy. Myocardial I/R was achieved by temporarily occluding the left coronary artery, then releasing the occlusion as previously described (11,12). The duration of ischemia was 30 minutes, and that of reperfusion 24 hours.
To delimit the ischemic area-at-risk (AAR) and area of necrosis (AN), Evans Blue 4% (Sigma-Aldrich Co^®, Missouri, USA) was injected through the left-ventricular apex. Thereafter, the heart sections were incubated with 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich Co^®, Missouri, USA) and fixed in 4% paraformaldehyde for 24 hours, as previously described [6 (link)]. The AN (white), AAR (not blue), and total left-ventricular (LV) areas from both sides of each section were measured using the software ImageJ 1.47. AAR/LV and AN/AAR were expressed as percentages, as previously reported [6 (link)].

Acetonitrile, hexane, acetone, methanol, and phenanthrene were obtained from Merck (Darmstadt, Germany). 2,3,5-Triphenyl tetrazolium chloride (TTC) and cadmium chloride were obtained from Sigma-Aldrich (analytical grade; San Luis, MO, USA). Bacterial growth media ingredients were purchased from BD Difco (Sparks, MD, USA). Reasoner 2A (R2A) and minimal growth media M9 were used for bacterial growth [45 (link)]. M9 was supplemented with anhydrous glucose or phenanthrene at 0.2% w/v.

Twenty-four hours after reperfusion, rats were anesthetized with an intraperitoneal injection of sodium pentobarbital and were intubated. The LAD was re-occluded, and 4% Evans blue dye (Sigma Aldrich) was injected via the inferior vena cava to identify area at risk (AAR). The heart was then excised and perfused with saline and cut into sequential 2-mm-thick cross sections. The sections were incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma Aldrich) for 10 minutes at 37°C and were photographed with a stereomicroscope (Nikon, HC-2500). The MI area (TTC negative, white), non-MI area within AAR (TTC positive/Evans blue negative, red), non-ischemic area (TTC positive/Evans blue positive, purple), and AAR (Evans blue negative) were analyzed using ImageJ software (version 1.44) (Fig 1).