Union International Contre le Cancer (UICC) chrysotile was sonicated (100 W, 30 s, Labsonic Sonicator; Sartorius Stedim Biotech S.A., Goettingen, Germany) before incubation with cell cultures to dissociate fiber bundles and to improve their suspension in the culture medium.
Labsonic sonicator
Manufactured by Sartorius
Sourced in France
The Labsonic sonicator is a laboratory equipment used for cell disruption and sample preparation. It utilizes high-frequency sound waves to break down cells and disperse samples. The Labsonic sonicator is a versatile tool for a range of applications in life science research and development.
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Lab products found in correlation
Optima l 90k ultracentrifuge, by Beckman Coulter (2 mentions)
Anti erk1 2, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Anti akt, by Merck Group (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence, by PerkinElmer (1 mentions)
Cholesterol fluorimetric assay kit, by Cayman Chemical (1 mentions)
Tissuelyser 2 device, by Qiagen (1 mentions)
Cholesterol cholesteryl ester quantitation assay kit, by Abcam (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Beas 2b, by American Type Culture Collection (1 mentions)
6 protocols using labsonic sonicator
1
Chrysotile Fiber Dissociation for Cell Cultures
2
Western Blot Analysis of Signaling Proteins
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After 24 hours of culture 2 × 106 cells were lysed in MLB buffer (125 mM Tris-HCl, 750 mM NaCl, 1% v/v NP40, 10% v/v glycerol, 50 mM MgCl2, 5 mM EDTA, 25 mM NaF, 1 mM NaVO4, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, pH 7.5), sonicated (with two bursts of 10 s; Labsonic sonicator, Sartorius Stedim Biotech S.A., Aubagne Cedex, France), and centrifuged at 13000 × g for 10 min at 4°C. Ten μg cell lysates were subjected to SDS-PAGE, transferred to polyvinylidene fluoride membrane sheets (Immobilon-P, Millipore, Billerica, MA, USA) and probed with the following antibodies: anti phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1–2 (Millipore); anti-ERK1–2 (Millipore); anti-phospho-(Ser473)-Akt (Millipore); anti-Akt (Millipore); anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH, used as control of equal protein loading; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), followed by the secondary peroxidase-conjugated antibodies (Bio-Rad). Proteins were detected by enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA).
3
Cholesterol Quantification in Tumor Homogenates
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Tumors were homogenized in 750 µL PBS using the Tissue Lyser II device (Qiagen), as per manufacturer’s instructions, then sonicated (two bursts of 10 s; Labsonic sonicator, Sartorius Stedim Biotech S.A., Aubagne Cedex, France). 500 µL were centrifuged at 13000 g for 15 min at 4 °C. The supernatants were centrifuged at 100000 g for 1 h at 4 °C, using a Optima L-90K Beckman Coulter Ultracentrifuge (Beckman Coulter Inc, Fullerton, CA) in lysis buffer (10 mM Tris, 100 mM NaCl, 20 mM KH2PO4, 30 mM EDTA, 1 mM EGTA, 250 mM sucrose, pH 7.5) supplemented with protease inhibitor cocktail set III, 1 mM Na3VO4, 1 mM NaF, 1 mM4-(2-aminoethyl)benzenesulfonyl fluoride (PMSF), 10 mM aprotinin and 10 mM dithiothreitol (DTT). The pellet (corresponding to microsomal fraction) was re-suspended in 500 µl lysis buffer and stored at −80 °C until use. 250 µl was used for the measurement of HMGCR activity, and 250 µL of tumor homogenates before the ultracentrifugation was used to measure total cholesterol. For both total and membrane-associated cholesterol, we used the Cholesterol Fluorimetric Assay kit (Cayman Chemical, Ann Arbor, MI) as per the manufacturer’s instructions. Results were expressed as µmol cholesterol/mg cell proteins.
4
Membrane Cholesterol Quantification Protocol
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10 × 106 cells were lysed in 0.5 mL of 10 mM Tris, 100 mM NaCl, 20 mM KH2PO4, 30 mM EDTA, 1 mM EGTA, 250 mM sucrose, pH 7.5 and sonicated with 2 bursts of 10 s (Labsonic sonicator, Sartorius Stedim Biotech S.A., Aubagne Cedex, France), then centrifuged at 13000 g for 15 min at 4 °C. The supernatants were centrifuged at 100000 g for 1 h at 4 °C, using an Optima L-90K Beckman Coulter Ultracentrifuge (Beckman Coulter Inc, Fullerton, CA) to collect the membrane fractions. The pellets were resuspended in 250 µL of the assay buffer provided by fluorimetric Cholesterol/Cholesteryl Ester Assay Kit – Quantitation (Abcam) and used to measure free cholesterol in the membrane, as per manufacturer’s instructions. An aliquot of 50 µl was sonicated again to measure the membrane proteins. Results were expressed as mg cholesterol/mg membrane proteins.
5
Asbestos Fiber Dissociation for Cell Cultures
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UICC (Union International Contre le Cancer) chrysotile and UICC crocidolite were sonicated (100 W, 30 sec, Labsonic Sonicator; Sartorius Stedim Biotech S.A.) before incubation with cell cultures to dissociate fiber bundles and to improve their suspension in the culture medium.
6
Immortalized BEAS-2B Cell Exposure to TiO2
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Human bronchial epithelial (BEAS-2B) cells are immortalized cells obtained from American Type Culture Collection (ATCC, Manassas, VA). They were cultured in 35-(1.2 × 10 6 cells) or 100-(7.5 × 10 6 cells) mmdiameter petri dishes in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified incubator at 37°C in a 5% CO 2 atmosphere.The generic cell culture protocol consisted of growing the cells in an incubator at 37°C 5% CO 2 in 75 or 150 cm 2 flasks, replacing media every 2-3 days, and passaging before confluence by dislodging with trypsin, washing and seeding new dishes or treatment wells. Prior to the commencement of the assay, TiO 2 samples (1-5-10 μg/cm 2 equal to 5-25-50 μg/mL) were sonicated (100 W: 30 sec; LabsonicSonicator; Sartorius Stedim Biotech S.A., Aubagne, France) to allow better suspension in the culture medium. Cells were incubated for 24, 48 or 72 h in the absence or presence of TiO 2 . All biological experiments were performed in triplicate (n=3).The protein content of the monolayers and cell lysates was assessed with the BCA kit from Pierce (Rockford, IL).
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