Using an MTT assay, cell viability was evaluated. The cells were seeded in 96-well plates at a density of 3×103 cells per well. Following incubation overnight, the cells were exposed to leptin (Sigma-Aldrich); for 24–96 h Following incubation overnight, the cells were exposed to different concentration of leptin (0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 µg/ml) for 24 h or the cells were exposed to 0.5 µg/ml leptin for 24–96 h. Fresh complete medium (0.2 ml) supplemented with 20 µl MTT dye [(5 mg/ml in phosphate-buffered saline (PBS)] was added to each well. Following incubation at 37°C for 4 h, each well was washed thoroughly with PBS. Subsequently, dimethyl sulfoxide (Sigma-Aldrich) was added to each well to terminate the MTT reaction and dissolve the formazan crystals. Following agitation at room temperature for ~10 min, the optical density (OD) of each well was measured at 450 nm. The viability of the control cells were considered as 100%, and the results were calculated as follows: (ODtreated cells − ODcontrol cells) / (ODcontrol cells − ODblank) × 100%.
Leptin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Canada, Italy, France, Japan
Leptin is a hormone produced by adipose (fat) tissue. It plays a role in regulating energy balance, appetite, and metabolism. Leptin lab equipment is used for the detection and quantification of leptin levels in biological samples.
Automatically generated - may contain errors
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132 protocols using leptin
1
Leptin Effects on Cell Viability
2
Immortalized Mouse Podocyte Culture
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The conditionally immortalized mouse podocyte cell line was provided by Professor Maria Pia Rastaldi (S. Carlo Hospital, University of Milan, Italy) and cultured as described previously.[12 ] Well-differentiated immortalized mouse podocytes were used for experiments. The podocytes were incubated with the RPMI-1640 medium with 5% fetal bovine serum and grouped as follows: medium alone; medium containing leptin (Abcam, UK); medium containing A438079 (Tocris Bioscience, UK) or KN-62 (Sigma-Aldrich, USA); and medium containing both leptin and A438079 or leptin and KN-62 (cells were preincubated with A438079 or KN-62 for 30 min, and then leptin was added in the culture medium). The concentrations of leptin, KN-62, and A438079 in the medium were 20 ng/ml, 2.5 μmol/L, and 10 μmol/L, respectively, of which all had no cytotoxic effects on podocytes confirmed by lactate dehydrogenase (LDH) release assay [Supplementary Material 1 ].[21 (link)]
3
Immunostimulant-Induced Cytokine Responses
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Mice (n = 8 per group) were immunized into the thigh muscles with 50 μL total volume (25 μL per thigh) with OVA (0.5 μg/total dose), poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP, 2.5 μg/dose, Idaho National Laboratory, Idaho Falls, ID, US), Curdlan (0.5 μg/total dose, Wako Pure Chemical Industry, Ltd. Japan)), Leptin (0.5 μg/dose, Millipore Sigma, Oakville, Ontario, Canada), PCEP+Curdlan, Curdlan+Leptin, PCEP+Curdlan+Leptin or phosphate-buffered saline (PBS) on day 0 and day 21 (booster). Vaccine diluent was PBS, pH 7.4 (Gibco, Life Technologies). Spleens were excised on day 35 and processed to a single-cell suspension. Cells were stimulated with OVA (1 μg/mL) or left unstimulated for 48 h then subjected to flow cytometric analysis to immunotype the T cell population that were induced in response to OVA.
Local cytokine production after intramuscular injection with immunostimulants: mice were injected with 50 μL total volume (25 μL per thigh) with PCEP (2.5 μg/dose), Curdlan (0.5 μg/dose), Leptin (0.5 μg/dose), PCEP+Curdlan, Curdlan+Leptin, or PBS into the thigh muscles. After 48 h, muscles were excised, processed into a single cell suspension, and cells were lysed (details below).
Local cytokine production after intramuscular injection with immunostimulants: mice were injected with 50 μL total volume (25 μL per thigh) with PCEP (2.5 μg/dose), Curdlan (0.5 μg/dose), Leptin (0.5 μg/dose), PCEP+Curdlan, Curdlan+Leptin, or PBS into the thigh muscles. After 48 h, muscles were excised, processed into a single cell suspension, and cells were lysed (details below).
4
Hormone and Metabolite Profiling Protocol
5
Leptin and Wnt Inhibitor Effects on Cell Proliferation
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Cells from each cell line were transferred into 96-well plates containing the corresponding aforementioned medium with 5×103 cells per well. Following incubation at 37°C for 3–5 h, cell adhesion was reached and 100 µl Dulbecco's modified Eagle's medium (Sigma-Aldrich; Merck KGaA) was added. Cells were cultured at 37°C with the different concentrations of leptin (0, 25, 50 and 100 mM; Sigma-Aldrich; Merck KGaA), and 10 µl cell counting kit-8 solution (Sigma-Aldrich; Merck KGaA) was added at 24, 48 72 and 96 h later. Following incubation for another 4 h, optical density values at 450 nm were measured using a microplate reader. For Wnt inhibitor PNU-74654 (20 µM; Sigma-Aldrich; Merck KGaA) treatment, 20 µM PNU-74654 and 100 mM leptin was added to the culture medium under the same conditions as aforementioned.
6
Serum Biomarkers in Murine EAE
7
Hormonal Regulation of Lymphocyte Function
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Lymphocytes were incubated with medium 199 (control) or with the following human hormones: adiponectin (Sigma, St. Louis, MO, USA) at 100 ng/mL, leptin (Thermo Fisher, Carlsbad, CA, USA) at 100 ng/mL, melatonin (Sigma, St. Louis, MO, USA) at 100 ng/mL, adiponectin + leptin at 100 ng/mL, and melatonin + leptin at 100 ng/mL. The stimuli were endotoxin-free. The hormone concentrations were determined according to previous research with mononuclear cells from human colostrum taken from eutrophic and obese puerperal women [6 (link),7 (link)].
8
Leptin Dose-Dependent Metabolic Effects
9
Adipokine Effects on MCF10A Cells
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MCF10A cells were purchased from ATCC (ATCC® CRL-10317TM) and maintained in DMEM/F12 supplemented as described by Brugge lab1 and elsewhere. The cells were subcultured before reaching 80% confluence. For adipokine treatments the MCF10A cells were serum-starved overnight (Yan et al., 2012 (link)). Leptin (#L4146) and adiponectin (#SRP4901) were purchased from Sigma-Aldrich and added to final concentrations of 50–100 ng/ml (Leptin) and 25–250 ng/ml (adiponectin). The cells were incubated with adipokines for duration 24–72 h. As mechanosensitive Ca2+ channel inhibitor was used GSMTx4 (#ab141871, Abcam) in concentration 2.5 μM for 16 h.
For siRNA silencing, 25 nM ON-TARGET plus SMARTpoolTM siRNA library AMPK(#J-005027-06 Dharmacon, GE Healthcare) or Ob-R (Leptin receptor, #L-008015-00-0005 Dharmacon, GE Healthcare) were transfected into cells on 35 mm plates by using RiboJuice transfection reagent (#71115, Novagen) according to the manufacturer’s instructions. Cells were incubated for 72–96 h for efficient depletion of the target protein.
For siRNA silencing, 25 nM ON-TARGET plus SMARTpoolTM siRNA library AMPK(#J-005027-06 Dharmacon, GE Healthcare) or Ob-R (Leptin receptor, #L-008015-00-0005 Dharmacon, GE Healthcare) were transfected into cells on 35 mm plates by using RiboJuice transfection reagent (#71115, Novagen) according to the manufacturer’s instructions. Cells were incubated for 72–96 h for efficient depletion of the target protein.
10
Leptin and FABP4 Regulation Study
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After transfection with FABP4 reconstructed vectors for 2 days, cells were washed with phosphate-buffered saline (PBS) twice. Then cells were then incubated in serum-free medium for 90 min before 100 nM leptin (Sigma, USA) was added. After 6 h, 12 h, 18 h, and 24 h cells were washed once with ice-cold PBS and then cells were harvested separately for further study. 1 μM Oligomycin (Sigma, USA) was performed to incubate cells for 1 h according to the same protocol as leptin.
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