Experiments were conducted on age- and gender-matched Ctns knockout mouse (C57BL/6 background4 (link)) or rat (Sprague-Dawley background22 (link)) lines, and their corresponding control littermates. Due to an effect of sex on cystine content (higher levels in female versus male kidneys mice5 (link),15 (link),43 (link)), only female mice at age 6, 12, and 24 weeks were used for primary cell cultures and mechanistic studies. Rats and mice were housed under specific pathogen free conditions and maintained under temperature (22–25 °C)- and humidity (50–60%)-controlled conditions with 12-h dark/light cycle with water and food provided ad libitum. Kidneys were collected for analyses at the time of sacrifice. Both male and female rats aged 12 weeks were subcutaneously implanted with a slow-release pellet (Innovative Research of America, FL, USA). The pellets were designed to allow a constant release of rapamycin (1.5 mg/kg B.W./day; R-5000, LC Laboratories, Woburn, MA). Control animals received a placebo pellet equivalent to their respective rapamycin-treated group. After 2 weeks of treatment, the rats were sacrificed, and the kidney tissues were harvested and collected for subsequent analyses.
R 5000
Manufactured by LC Laboratories
Sourced in United Kingdom
The R-5000 is a high-performance laboratory instrument designed for precise and efficient sample analysis. It features advanced technology and robust construction to ensure reliable and consistent results. The core function of the R-5000 is to provide accurate and repeatable measurements for a variety of applications in the scientific and research sectors.
Automatically generated - may contain errors
Lab products found in correlation
Rapamycin, by LC Laboratories (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fk506, by LC Laboratories (1 mentions)
Tacrolimus, by LC Laboratories (1 mentions)
Nephelostar galaxy, by BMG Labtech (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Pen strep, by Corning (1 mentions)
Microcal peaq itc analysis software, by Malvern Panalytical (1 mentions)
Microcal peaq itc, by Malvern Panalytical (1 mentions)
Torin1, by LC Laboratories (1 mentions)
C6628, by Merck Group (1 mentions)
M9281, by Merck Group (1 mentions)
G1264, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Baclofen, by Bio-Techne (1 mentions)
Ro25 6981, by Bio-Techne (1 mentions)
Cgp35348, by Bio-Techne (1 mentions)
Ro 25 6981, by LC Laboratories (1 mentions)
Bumetanide, by Merck Group (1 mentions)
10 protocols using r 5000
1
Cystinosis Mouse and Rat Models
2
Evaluating mTOR and CNi Modulators
3
Rapamycin Treatment Impacts Photoreceptor Survival
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In accordance with a previous report36 (link), wild-type and coa mutant embryos were treated with rapamycin (LC laboratories, R-5000) at 10 μM from 34 to 97 hpf, and fixed in 4% PFA. Photoreceptor survival was evaluated with rhodamine-conjugated phalloidin labeling. Using Image-J software, the photoreceptor cell layer was outlined and its size was determined. Means and standard deviations were calculated. Statistical analysis was done using two-way ANOVA with the Tukey multiple comparison test.
4
Nephelometric Analysis of Fungal Spore Germination
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Germination of P. chrysosporium RP78 conidia was measured using a nephelometric reader (NEPHELOstar Galaxy, BMG Labtech, Offenburg, Germany). For inocula, suspensions of spores were obtained from 8 day-old mycelia grown on sporulation medium (Glucose (10 g.L-1), malt extract (10 g.L-1), peptone from potato (2 g.L-1); yeast extract (2 g.L-1), asparagine (1 g.L-1), KH2PO4 (2 g.L-1), MgSO4.7H2O (1 g.L-1), thiamine HCl (1 mg.L-1), Agar (30 g.L-1), all chemicals used were purchased from Sigma Aldrich. Spores were collected with gentle scraping of the agar plates and filtration through Miracloth. The number of spores per 1 mL of suspension was determined with optical density (OD) measurement at 650 nm, and calculated as previously described [24 (link)]. For each microplate well, 200 μl of sample were prepared: 10,000 spores were resuspended in 198 μL of malt 1% and 2 μL of rapamycin (LC laboratories, R-5000) were added for treatment or 2 μl of DMSO as mock for control. Equipment was set up with the following parameters: temperature of incubation was 37°C, cycle time 1 hour and the sum of cycles was 72 hours. Relative nephelometric unit (NRU) values were calculated as previously described [25 (link)].
5
Inducible Chromatin Remodeling in Mouse ES Cells
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Mouse embryonic stem (ES) cells CiA:Oct4 infected with N118 and N163 plasmids (N118, nLV EF-1α-Gal-FKBPx1-HA-PGK-Blast; N163, nLV EF-1α-HP1α (CS)-Frbx2(Frb+FrbWobb)-V5-PGK-Puro) were grown in cell culture media containing high-glucose DMEM (Corning, 10013CV), 20% FBS(Gibco 26140–079), 10 mM HEPES pH 7.5(Corning, 25060Cl), NEAA(Gibco 11140050), Pen/Strep (Corning, 20002CL), 2-Mercaptoethanol(Gibco, 21985023), and 1:500 LIF conditioned media produced from Lif 1Ca (COS) cells. ES cells were cultured essentially as previously described (Hathaway et al., 2012 (link)). CiA:Oct4 with N118 and N163 were selected with puromycin (invivogen, ant-pr) and blasticidin (invivogen, ant-bl) for three days, and remove selection one day prior to rapamycin (LC laboratories, R-5000) treatment. For the time course experiment, CiA:Oct4 with N118 and N163 were plated in 80,000 cells per well in a 6-well plate for the time course experiment. Rapamycin from a 10 μM stock dissolved in ethanol was added to media at 3 nM concentration for the time course experiment. CiA:Oct4 cell lines were generated in a previous study (Hathaway et al., 2012 (link)). No animals were used in this work.
6
Thermodynamic Characterization of Rapa-FKBP Binding
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The drug binding interactions between Rapa (R-5000, LC Laboratories Inc., Woburn, MA) and FKBP-ELPs were studied using ITC (Figure 3 ) on a MicroCal PEAQ ITC (Malvern Instruments Ltd, Worcestershire, United Kingdom). The reference cell of the calorimeter was filled with water and all binding studies were performed at 37 °C. Reverse titrations were performed with a fixed Rapa concentration in the calorimeter cell and FKBP-ELPs in the titration syringe. The drug and all the FKBP-ELPs were solubilized in the same buffer (2.4% v/v DMSO in PBS) to prevent background heat of release due to differences in buffer composition. Briefly, 300 µl of 8 µM Rapa was carefully loaded into the calorimeter cell and a titration syringe filled with 50 µM FAF or 100 µM FA/ FSI was injected (3 µl) into the calorimeter cell 12 times. The resulting isotherm was fitted to a 'one set of sites' binding model in offset mode using the MicroCal PEAQ ITC analysis software (Malvern Instruments) to estimate affinity (Kd), stoichiometry and thermodynamics (ΔH, ΔS and ΔG).
7
Autophagy and GABA-A Receptor Modulation Assays
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Cells were treated with drugs or starvation media Earl’s Balanced Salt Solution (EBSS) (Thermo Fisher, 24010043) 24 h after plating in 35 mm dishes or 96-well plates (50% confluence) for 24 h (unless specified) in an incubator at 37 °C with 5% CO2. For astrocyte experiments, cells were treated 24 h after transfection under the same conditions described above. Drug treatments were at 1:1000 dilutions, using 0.1% DMSO as a vehicle control, and media removal and replacement as a control for starvation.
Drugs used were: rapamycin (LC—Laboratories, R-5000 or Tocris, 1292), Torin1 (LC—Laboratories, T7887), Bafilomycin A1 (MilliporeSigma Calbiochem, 19600-010UG), non-commercial NAS were custom synthesized in house. We used NAS at 1 and 10 µM as having been shown effective in previous literature, including autophagy7 (link) and GABA-A receptor modulation13 (link). Note that 10 µM of GABA-A receptor modulating NAS is supraphysiological and near the solubility limit for uncharged NAS.
Drugs used were: rapamycin (LC—Laboratories, R-5000 or Tocris, 1292), Torin1 (LC—Laboratories, T7887), Bafilomycin A1 (MilliporeSigma Calbiochem, 19600-010UG), non-commercial NAS were custom synthesized in house. We used NAS at 1 and 10 µM as having been shown effective in previous literature, including autophagy7 (link) and GABA-A receptor modulation13 (link). Note that 10 µM of GABA-A receptor modulating NAS is supraphysiological and near the solubility limit for uncharged NAS.
8
Cochlear Epithelial Cell Culture and Autophagy Modulation
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HEI-OC1 cells, conditionally immortalized cochlear epithelial cells, were maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco-BRL) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL, 16000-044) at 33 °C with 10% CO2. OC explants from Sprague-Dawley rats at postnatal day 7 were isolated as previously described39 (link). Explants were maintained in DMEM including 10% FBS and 0.06 mg/ml penicillin at 37 °C with 5% CO2. GM (Sigma-Aldrich, G1264) was prepared in sterile dH2O. RPM (LC Laboratories, R-5000) was prepared in dimethyl sulfoxide (DMSO). 3-MA (Sigma-Aldrich, M9281) and CQ (Sigma-Aldrich, C6628) were prepared in sterile dH2O. Controls for each test compound used the respective solvent. Hydrogen peroxide (H2O2; Sigma-Aldrich, 516813) was used at 1 mM as a positive control for autophagy.
9
In Vivo and In Vitro Pharmacological Assays
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For behavioral and in vivo experiments, animals received intraperitoneal (i.p.) injections of Ro-25-6981 (10 mg/kg, Tocris 1594) with or without CGP35348 (100 mg/kg; Tocris, 1245), or 0.9% physiological saline vehicle (0.01 ml/g, LabChem). Animals in the RIP-Seq experiment received either Ro-25-6981 (10 mg/kg) with or without rapamycin (6 mg/kg; LC Laboratories, #R-5000), or 0.9% physiological saline (200 μl). For in vitro experiments, Ro-25-6981 (10 µM), rapamycin (200 nM), and baclofen (50 µM; Tocris, #0796) were used. Vehicle controls cells were treated with water (vehicle for Ro-25-6981 and baclofen) and/or 0.1% DMSO (vehicle for rapamycin).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.08.25.266833 doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.08.25.266833 doi: bioRxiv preprint
10
Pharmacological Modulation of Cell Dynamics
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Concentrations of drugs were kept constant throughout the experiment. Cells were preincubated in DMEM with drugs at 37 °C (see below for drug concentrations and incubation time). DMEM was replaced by Leibovitz with drugs and CellMask or Flipper-TR. The osmotic shocks were applied as before except that the solution contained the same drug concentration. The following pharmacological inhibitors were preincubated as follows: 50 nM latrunculin A for 1 h (Sigma L5163), 200 nM jasplakinolide for 30 min (Enzo ALX-350-275), 5 µM nocodazole for 30 min (Sigma M1404), 1 µM taxol for 1h (Sigma T1912), 100 µM DCPIB for 30 min (Tocris 1540), 50 µM EIPA for 30 min (Tocris 3378), 100 µM bumetanide for 30 min (Sigma B3023), 250 nM Torin1 for 30 min (LC Laboratories T-7887), and 100 nM rapamycin for 30 min (LC Laboratories R-5000). All the drugs were diluted in DMSO.
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