Native mitochondria were labeled with MitoTracker Green FM with excitation/emission 490/516 nm at 37°C for 45 minutes and washed twice with sterile PBS (Thermo Fisher Scientific). Isolated mitochondria were labeled with pHrodo Red Succinimidyl Ester with excitation/emission 560/585 nm for 30 minutes at 4°C, washed twice with sterile PBS (Thermo Fisher Scientific), and then coincubated with H9c2 cells. The nucleus was labeled with NucBlue Live with excitation/emission 360/460 nm (Thermo Fisher Scientific). A time‐lapse microscopy was subsequently conducted with both fluorescence and phase contrast/DIC channels to capture the dynamic behavior of mitochondrial internalization (Figure 2 B; Video S1 ). Images were acquired either using a wide‐field (Olympus IX83; Olympus Corporation, Tokyo, Japan) or confocal microscope (Zeiss LSM 780; Zeiss Microscopy, Jena, Germany). The respective data were analyzed using Fiji (NIH, Bethesda, MD) and IMARIS software (Bitplane AG, Zurich, Switzerland).
Sterile pbs
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Sterile PBS is a balanced salt solution that maintains physiological pH and osmolarity. It is commonly used as a buffer and diluent in a variety of laboratory applications.
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Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (14 mentions)
Streptomycin, by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Karmali agar, by Thermo Fisher Scientific (6 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Harvard Bioscience (5 mentions)
Streptomycin, by Harvard Bioscience (5 mentions)
24 flat bottom well culture plates, by Thermo Fisher Scientific (5 mentions)
Facscanto 2 flow cytometer, by BD (5 mentions)
Thermotop shaker, by Eppendorf (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Selective karmali agar plates, by Thermo Fisher Scientific (4 mentions)
Karmali agar plates, by Thermo Fisher Scientific (4 mentions)
Porcine pancreatic elastase, by Merck Group (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor cocktail tablet, by Roche (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
189 protocols using sterile pbs
1
Mitochondrial Dynamics Visualization
2
Glycolipid-induced HaCaT cell death assessment
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To determine the distinct morphological pattern of HaCaT cell death induced following treatment with each glycolipid congener and SLES, treated cells were stained with AO and PI (Lee et al. 2015 (link)). Experiments were set up and cells treated as described in the previous sub-section “Cell morphology assessment ”. Following treatment, the cells were washed three times with sterile PBS (ThermoFisher Scientific, Loughborough, UK) to remove detached cells and subsequently incubated with a 1:1 ratio of 100 μg mL−1 AO and PI (Merck, Gillingham, UK) for 3 min. To remove excess stains, the cells were washed three times with prewarmed sterile PBS (ThermoFisher Scientific, Loughborough, UK) and the stained cells were immediately imaged at 200 × magnification using Eclipse TS100 fluorescence microscope (Nikon Europe B. V., Amsterdam, The Netherlands). The excitation and emission wavelengths for AO were 493 and 535 nm, and for PI, 535 and 614 nm, respectively.
3
Cryptococcus gattii Strain Preparation and Murine Infection
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R265 C. gattii strain (B serotype, VGII molecular genotype) was recovered from 25% glycerol stocks stored at −80 °C and cultured on Sabouraud dextrose medium at 30 °C with constant agitation (150 rpm) for 16–18 h. C. gattii yeast with a thin capsule (C. gattii-thin) were obtained after inoculation in YPD medium containing 0.5 M NaCl, as described by Ikeda-Dantsuji et al. (2015) (link), and incubated at 30 °C with constant agitation at 150 rpm for 20–24 h. Yeast cells were harvested by centrifugation at 7,600 × g for 10 min at 25 °C and washed three times with sterile PBS (Thermo Fisher Scientific, Waltham, MA, USA). C. gattii-thin was heat-inactivated at 70 °C for 1 h (HK-C. gattii-thin). The cell concentration was determined using a Neubauer chamber with Chinese ink. A fraction of the HK-C. gattii-thin yeast was spread on Sabouraud dextrose agar to confirm the heat inactivation.
C57BL/6 mice were infected with viable yeast of C. gattii that were cultured on Sabouraud dextrose medium at 30 °C with constant agitation (150 rpm) for 16–18 h. Yeast cells were harvested by centrifugation at 7,600 × g for 10 min at 25 °C and washed three times with sterile PBS (Thermo Fisher Scientific). Cell concentration was determined using a Neubauer chamber with Chinese ink.
C57BL/6 mice were infected with viable yeast of C. gattii that were cultured on Sabouraud dextrose medium at 30 °C with constant agitation (150 rpm) for 16–18 h. Yeast cells were harvested by centrifugation at 7,600 × g for 10 min at 25 °C and washed three times with sterile PBS (Thermo Fisher Scientific). Cell concentration was determined using a Neubauer chamber with Chinese ink.
4
Acridine Orange/Propidium Iodide Staining of Treated Cells
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To assess the morphological pattern of cell death induced by mono-RL and Piceatannol treated cells an acridine orange (AO)/propidium iodine (PI) staining technique was utilized [39 (link)]. HCT 116, Caco2 and CCD-841-CoN cells were cultured and treated as previously described in Section 2.7 . Following treatment, cells were washed three times with sterile PBS (Thermo Fisher Scientific) and subsequently incubated for 3 min with 100 μg mL−1 AO and PI (Merck Life Science, Bengaluru, India) mixed at a ratio of 1:1. Cells were then re-washed with sterile PBS (Thermo Fisher Scientific) and stained cells were imaged at 200× magnification using an Eclipse TS100 fluorescence microscope (Nikon). The excitation and emission wavelengths for AO were 493 nm and 535 nm, and for PI were 535 nm and 614 nm. All experiments were carried out in triplicate with three technical replicates per treatment condition. Each replicate was imaged three times and images for publication were randomly selected for publication using a computer. Scale bars (100 μm) were added to images using ImageJ 1.53t software [38 (link)].
5
Elastase-Induced Emphysema Model
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Emphysema was induced as described previously (Kneidinger et al., 2011 (link)), using elastase, an enzyme known to induce destruction of lung tissue. The elastase-induced injury model is widely used to study emphysema and well documented (Wright et al., 2008 (link)). Briefly, porcine pancreatic elastase (Sigma-Aldrich) was dissolved in sterile PBS (Gibco) and applied orotracheally, 40-U·kg−1 in 80-μl PBS, in C57BL/6J mice anaesthetized with 3% isoflurane . Animal health status and weights were closely monitored daily.
6
Murine Viral Infection and Protein Immunization
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All animal experiments were performed in accordance with institutional guidelines and Swiss federal regulations. Experiments were approved by the veterinary office of the canton of Zurich (animal experimentation permissions 115/2017) and Basel‐Stadt (animal experimentation permission 2582). A 6‐ week‐old female C57BL/6 mouse was infected with 2 × 106 LCMV clone 13 intravenously as previously described [13 (link)] and sacrificed at 28 dpi. LCMV clone 13 was propagated by infection of BHK‐21 cells for 24–48 h and then resuspended in sterile PBS (GIBCO).
For protein immunizations, a 6‐week‐old male C57BL/6 mouse was repeatedly immunized five times every other week s.c. into the flank with 10 μg of human TNFR2 protein (Peprotech, 310–12)/20 μg MPLA (Sigma, L6895) adjuvant and sacrificed 1 week afterwards. Likewise, a 6‐week‐old female BALB/c mouse was injected three times with 4 and 3 weeks intervals s.c. into the flank with 100 μg of OVA (Sigma, A5503)/20 μg MPLA (Sigma, L6895) adjuvant and sacrificed 2 weeks after the final boost.
For protein immunizations, a 6‐week‐old male C57BL/6 mouse was repeatedly immunized five times every other week s.c. into the flank with 10 μg of human TNFR2 protein (Peprotech, 310–12)/20 μg MPLA (Sigma, L6895) adjuvant and sacrificed 1 week afterwards. Likewise, a 6‐week‐old female BALB/c mouse was injected three times with 4 and 3 weeks intervals s.c. into the flank with 100 μg of OVA (Sigma, A5503)/20 μg MPLA (Sigma, L6895) adjuvant and sacrificed 2 weeks after the final boost.
7
Antioxidant Assays in Yeast Cells
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Glucose and bovine serum albumin (BSA) were provided by China National Pharmaceutical Group. Saccharomyces cerevisiae was supplied by Angel Yeast Co., Ltd (Yichang, China). 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), 2,4,6-tri(2-pyridyl)-1,3,5-trianzine (TPTZ), ferric chloride hexahydrate, trichloroacetic acid, ethanol, and dimethyl sulfoxide were purchased from Sinopharm Chemical. Catechin, epicatechin and p-Coumaric acid were bought from Maclean Biotech. Non-essential amino acids and minimum essential medium (MEM) were made available from Hyclone. Fetal bovine serum and sterile PBS were obtained from Gibco. dimethyl sulfoxide was carried out by Sigma. 0.25% trypsin-EDTA were purchased from Jinuo Biotechnology (Hangzhou, China).
8
Quantifying Cellular Uptake of Cy3-SWCNTs
9
Unilateral Injection of Preformed Fibrils in Olfactory Bulb
10
Bronchoalveolar Lavage Fluid Collection
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Mice were euthanized by intraperitoneal injection of 75 mg/kg sodium pentobarbital (Nembutal; Abbott Labs, Chicago, IL) as previously described by Elliott et al.27 (link). Tracheas were exposed, nicked at the bottom of the larynx and cannulated. The proximal ends of the tracheas were tied off and 1.0 mL of cold sterile PBS (Gibco, Grand Island, NY) was gently flushed into the lungs and recovered; a total of three washes were performed and combined. Collected BALF was centrifuged at 300 g for 7 min at 4 °C. The supernatant was stored at −80 °C until use for measurement of cytokines concentration. Pelleted cells were resuspended in 1.0 ml of PBS. Total cell were counted on a hemocytometer, and 1–5 × 103 cells were spun onto glass microscope slides (cytospin 3; Shandon Scientific, Cheshire, UK). Cells were air dried for 24–36 h, fixed, and stained with a Diff-Quik stain set (Dade Behring, Newark, DE). Differential cell counts of at least 300 cells per slide were made according to morphological criteria. The number of cells recovered was calculated and expressed as absolute cell numbers.
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