Wild-type S2 cells (1.5×106) were plated on coverslips and permeabilized with 10 µg/ml digitonin (D141, Sigma-Aldrich) in KRB for 2 h at 26°C. Subsequently, cells were fixed and Sec bodies were visualized by immunostaining of Sec16. To test the effect of ATP, AMP or adenosine on Sec body formation, the semi-intact cells were incubated in the presence of 0.5 mM ATP (A1852, Sigma-Aldrich), 0.5 mM AMP (01930, Sigma- Aldrich) or 0.5 mM adenosine (A9251, Sigma-Aldrich). Note that the digitonin was not removed. The permeabilization efficiency was determined by using the non-membrane-permeable dye TO-PRO-3 iodide (T3605, Thermo Fisher Scientific) (Fig. 6 A). The import buffer used in the SIC system was 20 mM HEPES, 110 mM KAc, 2 mM MgAc, 5 mM NaAc and 0.5 mM EGTA (pH was set at either 6.0 or 7.4 with KOH). For the RNA degradation experiment in the SIC system, we incubated S2 cells on coverslips for 2 h at 26°C in Schneider's medium containing 10 µg/ml digitonin (Sigma-Aldrich) with or without 0.25 U/ul RNase 1 (EN0602, Thermo Fisher Scientific).
A1852
Manufactured by Merck Group
Sourced in United Kingdom, Sweden
A1852 is a laboratory equipment product. It is a scientific instrument designed for conducting various experiments and analyses in a research or laboratory setting. The core function of A1852 is to perform specific tasks related to the investigation and examination of samples or materials, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
K gluconate, by Merck Group (2 mentions)
K885300 0002, by Thermo Fisher Scientific (2 mentions)
Digitonin, by Merck Group (1 mentions)
A9251, by Merck Group (1 mentions)
Rnase 1, by Thermo Fisher Scientific (1 mentions)
Fura red am, by Thermo Fisher Scientific (1 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
F3917, by Merck Group (1 mentions)
Easymount chamber, by Physiologic Instruments (1 mentions)
A7410, by Merck Group (1 mentions)
Ab142780, by Abcam (1 mentions)
T9033, by Merck Group (1 mentions)
Ab120124, by Abcam (1 mentions)
Tcs sp5, by Leica (1 mentions)
Celltiter glo, by Promega (1 mentions)
N suc llvy amc, by Merck Group (1 mentions)
Nonidet p 40, by Merck Group (1 mentions)
Enspire, by PerkinElmer (1 mentions)
N8129, by Merck Group (1 mentions)
G7021, by Merck Group (1 mentions)
14 protocols using a1852
1
Sec Body Formation Regulation by Nucleotides
2
Calcium Imaging of SCG Neurons
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Cultured SCG neurons on day 7 were used to perform Ca2+ imaging. Neurons were washed with Hank's balanced salt solution without Ca2+ and Mg2+ three times every five minutes and then covered with a sufficient amount of Fura‐Red AM (10 μm, F3021, Invitrogen) with PluronicF‐127 (P6867, Invitrogen) in Hank's balanced salt solution without Ca2+ and Mg2+ at 37°C for at least 60 minutes. Cells were incubated for another 30 minutes with Hank's balanced salt solution with Ca2+ and Mg2+. Images were immediately stored on a high‐speed hard drive. The ratio of fluorescence intensity changes excited at 488 nm and collected at 625‐725 nm was computed as previously reported.57 , 58 , 59 The formula for calculating fluorescence intensity changes is ΔF/F = (F‐Fbase)/(Fbase‐B), where F indicates the real fluorescence intensity, Fbase is the intracellular fluorescence intensity before stimuli, and B is the background fluorescence intensity. Then, ATP (10 µM, A1852, Sigma‐Aldrich), muscimol (10 µM, G019, Sigma‐Aldrich) or (and) SR95531 (1 µM, S106, Sigma‐Aldrich) were added to the extracellular fluid to measure the ratio of fluorescence intensity at 488 nm. Pictures were taken every 1 minute.
3
Ussing Chamber Analysis of HBECs
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Fully differentiated HBECs on Snapwell filters were mounted in Ussing chambers (Easymount chamber; Physiologic Instruments) connected to a VCC MC6 voltage clamp unit (Physiologic Instruments, San Diego, CA, USA). Solutions were maintained at 37 °C by heated water jackets and bubbled with air. For BK activity, basolateral membranes were permeabilized for 30 min with 20 µM amphotericin B, 10 µM nigericin and 10 µM valinomycin because whole cell short circuit current recordings cannot distinguish K+ efflux (it measures net current, a combination of K+ and Cl− efflux)10 (link). Furthermore, cells were exposed to a basolateral (140 mM) to apical (5 mM) K+ gradient, in the presence of apically applied 10 µM amiloride (Sigma-Aldrich #A7410, St. Louis, MO, USA) and 10 µM ATP (Sigma-aldrich #A1852). CFTR activity was measured with apical 5 mM Cl− in the presence of apically applied 10 µM amiloride, 10 µM forskolin (Sigma-Aldrich #F3917) followed by 10 µM CFTRinh172 (Sigma-Aldrich #C2992)8 (link).
4
Rearing and Feeding of Aedes aegypti
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Aedes aegypti were reared and kept in an environmental room under LD 12:12 h cycle at 26 −28°C, 79% RH. Eggs were hatched by adding deoxygenated water with ground fish food (catalog #16152, Tetra, Melle, Germany) inside a plastic container (L: 32 × W: 17 × H: 10 cm). Post-hatching, larvae were fed daily with ground fish food. The pupae were placed in small cups with distilled water and moved to a mesh cage (L: 30 × W: 30 × H: 30 cm DP100B, Bugdorm store, Taiwan), and allowed to eclose. Adult mosquitoes were fed on 10% sucrose solution (weight: volume in distilled water) from a cotton wick inserted into a vial. Mosquitoes were blood-fed using an artificial blood feeder (CG-1836, Chemglass Life Sciences, USA) filled with defibrinated sheep blood (SB055, TCS Biosciences Ltd, Buckingham, UK) (heated to 37°C), spiked with 10 mM ATP (A1852, Sigma-Aldrich) for about 2 hours per cage. Blood-fed mosquitoes were subsequently allowed to feed on 10% sucrose solution.
5
Lysosome Isolation and Immunoprecipitation
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Lysosome immunoprecipitation assay was conducted as a previously described protocol [70 (link)]. Briefly, Control and knockdown of MEF2A and MEF2D HeLa cells in a 15 cm dish were transfected for 48 h with Flag-tagged TMEM192 then performed amino acid starvation for 60 min and restimulation for 20 min. Cells were quickly rinsed twice and scraped in ice-cold KPBS (136 mM KCl, 10 mM KH2PO4, pH 7.25 was adjusted with KOH). Cells were collected under centrifuged at 1,000 g for 2 min at 4°C and then resuspended in 950 μl fractionation buffer (50 mM KCl, 90 mM K-gluconate [Sigma-Aldrich, 1550001], 1 mM EGTA, 5 mM MgCl2, 50 mM sucrose [Sigma-Aldrich, 573113], 5 mM glucose, 20 mM HEPES, pH 7.4, 2.5 mM ATP [Sigma-Aldrich, A1852], as previously described [27 (link)]) supplement with protease inhibitor. 50 μl content was reserved for further use as total cell lysis. Cells were physically broken using a 2 ml homogenizer (Thermo Fisher Scientific, K8853000002) and then centrifuged at 1,000 g for 2 min at 4°C. The supernatant containing the cellular organelles was mixed with 150 μL anti-Flag magnetic beads (Sigma-Aldrich, M8823) on a gentle rotator for 10 min at 4°C to enrich lysosomes. Immunoprecipitates with 1× SDS loading buffer were denatured for 5 min at 95°C and then analyzed by immunoblotting.
6
Rearing and Feeding of Aedes aegypti
7
Measuring Mitochondrial Calcium Dynamics
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Cardiomyocytes were seeded on glass-bottomed cell culture dishes and incubated with 1 μM of the calcium indicator Rhod2-AM (ab142780, Abcam) at 37°C in the dark for 30 minutes, as per the manufacturer’s guidelines. Subsequently, the cells were washed twice with calcium-free HBSS and imaged under a LSCM, Leica TCS-SP5). The fluorescence intensity (F) was normalized to the baseline fluorescence value F0 (F/F0) and expressed as mitochondrial calcium concentration ([Ca2+]m). We measured Fmax and Fmin, as previously described. Fmax was obtained by perfusion with 10-μM ionomycin and 5-mM CaCl2; Fmin was measured by perfusion with 10-mM ethylene glycol-bis (β-aminoethyl ether) -N,N,N′,N′-tetraacetic acid (EGTA) and 20-μM 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM; B1205, Molecular probes) in HBSS. Further, 2-aminoethoxydiphenyl borate (2-APB; ab120124, Abcam), TG (T9033, Sigma-Aldrich), and adenosine triphosphate (ATP; A1852, Sigma-Aldrich) were added to the external solution at a proper final concentration. The fluorescence intensity of Rhod2-AM was measured using LSCM. The fluorescence intensity was converted to [Ca2+] using the following formula: [Ca2+]m = Kd × (F − Fmin) / (Fmax − F), where Kd is the equilibrium dissociation constant of Rhod2 for Ca2+, which was 570 nM.
8
Quantifying ATP Levels in hTERT-BJ1 Cells
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ATP content was measured using CellTiter-Glo (G7570, Promega) and ATP standards (A1852, Sigma). 2 × 104 hTERT-BJ1 cells were seeded in black-walled 96 well plates. When cells were attached, drug treatments were added for 24, 48 or 72 h. Six replicates were used for each condition. Media was removed and CellTiter-Glo Assay was performed according to manufacturer's instructions. Light signal was acquired in the Xenogen VivoVision IVIS Lumina (Caliper Life Sciences). Results were normalised by SRB staining. ATP levels were calculated by extrapolating from the standard curve.
9
Mosquito Feeding Preference Analysis
10
Lysosome Isolation and Immunoprecipitation
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Lysosome immunoprecipitation assay was conducted as a previously described protocol [70 (link)]. Briefly, Control and knockdown of MEF2A and MEF2D HeLa cells in a 15 cm dish were transfected for 48 h with Flag-tagged TMEM192 then performed amino acid starvation for 60 min and restimulation for 20 min. Cells were quickly rinsed twice and scraped in ice-cold KPBS (136 mM KCl, 10 mM KH2PO4, pH 7.25 was adjusted with KOH). Cells were collected under centrifuged at 1,000 g for 2 min at 4°C and then resuspended in 950 μl fractionation buffer (50 mM KCl, 90 mM K-gluconate [Sigma-Aldrich, 1550001], 1 mM EGTA, 5 mM MgCl2, 50 mM sucrose [Sigma-Aldrich, 573113], 5 mM glucose, 20 mM HEPES, pH 7.4, 2.5 mM ATP [Sigma-Aldrich, A1852], as previously described [27 (link)]) supplement with protease inhibitor. 50 μl content was reserved for further use as total cell lysis. Cells were physically broken using a 2 ml homogenizer (Thermo Fisher Scientific, K8853000002) and then centrifuged at 1,000 g for 2 min at 4°C. The supernatant containing the cellular organelles was mixed with 150 μL anti-Flag magnetic beads (Sigma-Aldrich, M8823) on a gentle rotator for 10 min at 4°C to enrich lysosomes. Immunoprecipitates with 1× SDS loading buffer were denatured for 5 min at 95°C and then analyzed by immunoblotting.
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