ROS are formed in mitochondria and to high extent also in other cellular compartments of lung samples [24 (link)]. Therefore, total cytoplasmic protein was isolated from lung tissue in ice-cold 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (5 μl∙mg− 1 wet tissue) containing 1 μg∙ml− 1 saponine (Sigma, Saint Louis, MO) as mild detergent and 50 μM deferoxamine mesylate (Sigma) as Fe3+ chelator. Nuclei were then removed by centrifugation (600 g for 10 min). 10 μl nuclei-free cytoplasmic fraction were mixed after 90 min (Fe3+ chelate effect completed) with 90 μl 1 mM 1-hydroxy-3-carboxy-2,2,5,5,-tetramethyl-pyrrolidine (CPH; Noxygen, Elzach, Germany) as a spin trap for O2•-. A computer-operated electron paramagnetic resonance spectrophotometer (MiniScope MS100; Magnettech, Berlin, Germany) equipped with MiniScope Control v2.7.3 analysis software (Microtech GmbH, Bad Kreuznach, Germany) measured the time-dependent formation of 3-carboxy-2,2,5,5-tetra-methyl-pyrrolin-1-oxyl (•CP) at room temperature every 10 min. O2•- data were expressed as •CP formation rate∙min− 1.
Saponine
Manufactured by Merck Group
Sourced in United States
Saponine is a laboratory product manufactured by Merck Group. It is a natural surfactant derived from plants. Saponine reduces surface tension in aqueous solutions, facilitating wetting and emulsification.
Automatically generated - may contain errors
Lab products found in correlation
Deferoxamine mesylate, by Merck Group (1 mentions)
Fv1000 scanning confocal microscope, by Olympus (1 mentions)
Rabbit anti occludin, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 secondary antibody, by Jackson ImmunoResearch (1 mentions)
Pbs 1, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by GE Healthcare (1 mentions)
Tcs sp2 confocal microscope, by Leica (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Bismaleimide, by Thermo Fisher Scientific (1 mentions)
Malic acid, by Merck Group (1 mentions)
Succinic acid, by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Y 27632, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Dmi6000b fluorescence microscope, by Leica (1 mentions)
10 protocols using saponine
1
Measuring Cytoplasmic Superoxide Radical
2
Immunofluorescence Assay for Lipid Signaling
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Forty thousand cells were seeded on non-fluorescent gelatin-coated coverslips and allowed to adhere for 30 min. Following stimulations, as indicated in figure legends, cells were fixed with 2% paraformaldehyde in PBS for 10 min at room temperature. Where indicated, cells were permeabilized with 0.05% saponine (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 20 min and blocked with 2% BSA in PBS for 30 min. Then, cells were incubated with the appropriate primary antibodies for 2 h, and secondary antibodies for 1h or fluorescent phalloidin for 45 min, as indicated within the Figure legends. Images were taken with a FV1000 scanning confocal microscope (Olympus, Tokyo, Japan) coupled to an inverted microscope using a 63× oil immersion objective. For quantification of ATX/LPP or ATX/LPAR1 co-localization, cells were incubated with ATX and LPP1, LPP2, LPP3, or LPAR1 antibodies. The percentage of co-localization was calculated as previously described from serial optical sections of the whole cell [78 (link)].
3
Immunostaining of Tight Junctions in MEC Monolayers
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The MEC monolayers were washed three times with PBS 1× (Life Technologies) and gently dissociated from the insert filters followed by a 20-min fixation in paraformaldehyde (PFA) 4 % (w/v) (Sigma Aldrich, Saint-Louis, MO, USA) prior to immunocytochemistry. After three washes with PBS 1× (Life Technologies), the cells were pre-incubated for 30 min at room temperature (RT) with blocking buffer containing BSA 3 % (PAA Laboratories, Velizy-Villacoublay, France) in PBS 1×. The MEC monolayers were stained for 1 h in PBS 1× containing BSA 1 % (PAA Laboratories), with saponine 0.1 % (Sigma Aldrich) for membrane permeabilization, with a rabbit anti-occludin 1.5 μg/mL (Life Technologies). Cell nuclei were labeled with Hoechst 33342 1/1000 (Life Technologies) in co-incubation with a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Jackson Immunoresearch, West Grove, PA, USA). Cells were washed and mounted in Prolong Gold antifade mounting medium (Life Technologies). The mounted slides were observed with a Leica TCS SP2 confocal microscope (Leica Microsystems, Heidelberg, Germany). High-magnification images were acquired using a 63X HCX PL APO oil immersion objective and analyzed using the NIH ImageJ software (version 1.49o for Mac).
4
Bax Oligomerization Quantification by Flow Cytometry
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After fixing using 4% paraformaldehyde for 30 minutes, and cells were incubated for 1 hour at 4°C with the Bax-NT antibody (1:500; BD Biosciences) in PBS/1% FCS (Gibco, Carlsbad, CA, USA) + 0.1% saponine (Sigma Chemical Co.). After incubation with the secondary antibody, washing and re-suspension in PBS/1% FCS, and cells were measured by flow cytometry. For Bax oligomerization, the cells were suspended by conjugation buffer with 10 mM EDTA containing 0.2 mM bismaleimide (Thermo Scientific, Hudson, NH, USA) at room temperature for 1 hour and then extracted by lysis buffer for Western blot analysis.
5
Mitochondrial Respiration Profiling in Intestinal Epithelial Cells
6
Immunofluorescence Analysis of IDE in HeLa Cells
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Three days after siRNA transfection, 1×105 HeLa cells were cultured on 0.033% poly-L-lysine-treated 12 mm glass coverslips. Cells were fixed with 4% paraformaldehyde in PBS and quenched with 100 mM glycine, washed in PBS, treated with 0.2% BSA, 0.05% saponine (Sigma) and incubated for 45 min with 5 µg/ml of anti-IDE mAb. After washing, cells were incubated for 45 min with 10 µg/ml FITC-labeled goat anti-mouse antibodies (Abs; Jackson), washed again, fixed with 2% paraformaldehyde followed by glycine quenching and mounted with Fluoprep (BioMérieux). Images were taken with a DMI 6000B fluorescence microscope (Leica, Rueil-Malmaison, France) equipped with a 63x PlanApo objective and analyzed by 3D deconvolution using Metamorph®6.2 software (Universal Imaging Corp., Downington, PA).
7
Cell Cycle and Surface Marker Analysis
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MKN45 cells were seeded in 6-well plates 48 h before analysis. For cell cycle synchronization, cells were cultured in serum starvation for 24 h before replacing the serum in the media. On the day of analysis, cells were detached using Gibco® Versene solution (ThermoFisher, MA, USA) on indicated timepoints, spun down at 300 g for 5 min at 4 °C, and resuspended in Fixable Viability Dye APC-Cy7 (FVD 1:2000, ThermoFisher). For permeabilization, cells were fixed with 2% PFA and then treated with 0.5% Saponine (Sigma-Aldrich). For cell cycle analysis, cells were fixed in 70% ethanol. Afterwards, they were stained with primary antibodies CD44 (1:100), CD44v9 (1:100), or STn (undiluted) for 30 min on ice. Cells were washed and incubated with secondary antibodies anti-mouse or anti-rat (1:200) for 30 min on ice in the dark. For the cell cycle analysis, cells were stained with DAPI (10 μg/mL) for 20 min at room temperature in the dark. Samples were analyzed on a FACS Canto II Flow cytometer (BD, San Diego, CA, USA).
8
Quantifying Protein Synthesis via HPG Labeling
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To analyze protein synthesis activity, cellular incorporation of L-homopropargylglycine (HPG), a methionine analogue with alkyne moiety was detected by Click-iT reaction with azide-modified Alexa 48840 (link). Cells treated with HHT, imatinib or sunitinib for 1 h or 8 h were labelled with 25 µM HPG (Invitrogen, C10102) for the last 30 min of the drug treatment in L-methionine-depleted medium containing 5% dialyzed fetal calf serum (FCS; Gibco). Harvested cells were washed with PBS, fixed in 10% buffered formalin (4% formaldehyde) for 5 min at room temperature and permeabilized for 30 min in PBS containing 0.1% saponine (Sigma) and 1% FBS (HyClone). The Click-iT reaction was performed for 30 min at room temperature by suspending the cells in Click-iT cell reaction buffer containing 1 mM azide-modified Alexa 488 and 2 mM CuSO4 (Invitrogen). Cells were washed twice in PBS with 1% FCS, and the cell suspension was subjected to flowcytometry analysis. The protein synthesis inhibitor cycloheximide (100 µg/ml) was used as a positive control in this assay. Cells without Click-iT reaction were used as a background control.
9
Immunofluorescent Imaging of YAP and TAZ
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MDA-MB-231 cells were grown on 0.17 ± 0.01 mm thickness coverslips (Glaswarenfabik Karl Hecht GmbH and Co.). The cells were fixed with 3% formaldehyde (Fluka) for 5 min at 37 °C, then fixed again with 0.5% formaldehyde for 30 min at room temperature. The cells were washed with PBS and incubated in a quenching solution (50 mM NH4Cl in PBS) for 30 min at 4 °C. The cells were washed in PBS for 10 min and permeabilized with 0.2% Triton X-100 (Biotech) or 0.3% saponine (Sigma) in PBS containing 0.1% bovine serum albumin (BSA). Mouse anti-YAP1 antibody (1:50) or rabbit anti-TAZ antibody (1:50) was applied for 2 h to detect endogenous YAP or TAZ, followed by rinsing with PBS containing 0.1% BSA two times for 5 min. The cells were incubated with Alexa Fluor 488-conjugated secondary antibodies (1:500) (Invitrogen) for 1 h. After rinsing twice with PBS containing 0.1% BSA, the cells were mounted on a glass slide with Mowiol containing 4′, 6-diamidino-2-phenylindole. Images were collected on a Zeiss LSM 700 confocal microscope (Carl Zeiss) and analyzed with Zeiss ZEN software.
10
Cytochrome C Release Quantification
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Analysis of cytochrome C release was performed as previously described.43 (link) Briefly, cells were incubated in a digitonin-containing buffer for 30 min on ice (0.2 mM Na-EGTA, 100 mM KCl, 50 μg/ml digitonin, PBS), fixed with 4% PFA for 20 min and unspecific binding was blocked (3% BSA, 0.05% Saponine (Sigma-Aldrich), 1 h, RT) before incubation with cytochrome C antibody overnight at 4°C and staining with the secondary antibody for 1 h. Cytochrome C antibody was purchased from Cell Signaling Technology (Danvers, MA, USA) and secondary goat-anti-rabbit-Alexa-488 antibody was obtained from Invitrogen (Darmstadt, Germany). Fluorescence was detected using a FACSCalibur cytometer (Becton Dickinson). A decrease in fluorescence intensity indicates a loss of mitochondrial cytochrome C that is washed out after cell membrane permeabilization.
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