Proliferation was evaluated by CellTiter-Glo (Promega) starting from 2 × 103 HCC827 cells seeded on 96-well plates and incubated in the presence/absence of the indicated drugs for up to 7 days. Relative percentages were calculated by setting at 100% the average value of each curve on day 0. For anchorage-independent cell growth assay, cells were suspended in 0.45% type VII low-melting agarose (Sigma-Aldrich) in 10% FBS medium at a density of 6 × 104 cells/well, plated on a layer of 0.9% agarose in 10% FBS medium in 6-well plates and cultured for two weeks at 37 °C with 5% CO2. Relative percentages were calculated by setting at 100% the average colony counts in DMSO. Images were acquired with a Leica DMIRE2 microscope (Leica Microsystems). For drug sensitivity (IC50) experiments, 2 × 103 HCC827 cells were seeded on 96-well plates in the presence of serial dilutions of cetuximab, afatinib or erlotinib (Selleckchem). After incubation at 37 °C in 5% CO2, CellTiter-Glo (Promega) was used to measure luminescence according to the manufacturer’s protocol. Results were analyzed and plotted using GraphPad Prism version 9 software.
Low melting agarose type 7
Manufactured by Merck Group
Sourced in United States
Low-melting agarose (type VII) is a type of agarose designed for applications requiring low-melting temperatures. It is a natural gelling agent extracted from red seaweed. This agarose product has a lower gelling and melting temperature range compared to standard agarose, making it suitable for applications where heat-sensitive samples or components need to be protected.
Automatically generated - may contain errors
Lab products found in correlation
Dmire2 microscope, by Leica (2 mentions)
Celltiter glo, by Promega (2 mentions)
Cetuximab, by Selleck Chemicals (2 mentions)
Afatinib, by Selleck Chemicals (2 mentions)
Erlotinib, by Selleck Chemicals (2 mentions)
Mivacron, by GlaxoSmithKline (2 mentions)
S mem, by Merck Group (1 mentions)
Lr white resin, by Polysciences (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
5 protocols using low melting agarose type 7
1
Cell Proliferation and Colony Formation Assays
2
Cell Proliferation and Colony Formation Assays
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Proliferation was evaluated by CellTiter-Glo (Promega) starting from 2 × 103 HCC827 cells seeded on 96-well plates and incubated in the presence/absence of the indicated drugs for up to 7 days. Relative percentages were calculated by setting at 100% the average value of each curve on day 0. For anchorage-independent cell growth assay, cells were suspended in 0.45% type VII low-melting agarose (Sigma-Aldrich) in 10% FBS medium at a density of 6 × 104 cells/well, plated on a layer of 0.9% agarose in 10% FBS medium in 6-well plates and cultured for two weeks at 37 °C with 5% CO2. Relative percentages were calculated by setting at 100% the average colony counts in DMSO. Images were acquired with a Leica DMIRE2 microscope (Leica Microsystems). For drug sensitivity (IC50) experiments, 2 × 103 HCC827 cells were seeded on 96-well plates in the presence of serial dilutions of cetuximab, afatinib or erlotinib (Selleckchem). After incubation at 37 °C in 5% CO2, CellTiter-Glo (Promega) was used to measure luminescence according to the manufacturer’s protocol. Results were analyzed and plotted using GraphPad Prism version 9 software.
3
Imaging Transgenic Zebrafish Larvae Neuronal Activity
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Adult zebrafish (Danio rerio) were maintained and bred under standard conditions at 26.5°C. Embryos and larvae of a double-transgenic line (elavl3:GCaMP5 x vglut:DsRed)45 (link),46 (link) in nacre background were raised at 28.5°C in standard E3 medium47 .
Imaging experiments were performed as described previously48 (link),49 (link). In brief, larvae 4 - 5 days post fertilization were contained in a small drop of aerated E3 without methylene blue or N-phenylthiourea. Larvae were then paralyzed by addition of 20 μl of fresh mivacurium chloride (Mivacron, GlaxoSmithKline, Munich, Germany)50 (link) and embedded in 2% low-melting agarose (type VII; Sigma, St Louis, MO, USA) in a perfusion chamber that was inclined by 30° to improve dorsal optical access to the OBs. Agarose covering the noses was carefully removed. A constant stream of E3 (2 ml/min) was delivered through a tube in front of the nose and removed by continuous suction. Throughout the experiment it was ensured that larvae showed normal heartbeat. Larvae that were not fixed for EM recovered from paralysis after a few hours and continued to develop without obvious defects. All animal procedures were performed in accordance with official animal care guidelines and approved by the Veterinary Department of the Canton of Basel-Stadt (Switzerland). The sex of zebrafish larvae is not yet determined at the age used in this study.
Imaging experiments were performed as described previously48 (link),49 (link). In brief, larvae 4 - 5 days post fertilization were contained in a small drop of aerated E3 without methylene blue or N-phenylthiourea. Larvae were then paralyzed by addition of 20 μl of fresh mivacurium chloride (Mivacron, GlaxoSmithKline, Munich, Germany)50 (link) and embedded in 2% low-melting agarose (type VII; Sigma, St Louis, MO, USA) in a perfusion chamber that was inclined by 30° to improve dorsal optical access to the OBs. Agarose covering the noses was carefully removed. A constant stream of E3 (2 ml/min) was delivered through a tube in front of the nose and removed by continuous suction. Throughout the experiment it was ensured that larvae showed normal heartbeat. Larvae that were not fixed for EM recovered from paralysis after a few hours and continued to develop without obvious defects. All animal procedures were performed in accordance with official animal care guidelines and approved by the Veterinary Department of the Canton of Basel-Stadt (Switzerland). The sex of zebrafish larvae is not yet determined at the age used in this study.
4
Imaging Transgenic Zebrafish Larvae Neuronal Activity
5
Fixation and Embedding of HeLa Cells
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HeLa cells were cultured in a suspension in 150 cm2 tissue culture flasks (Techno Plastic Products AG, Trasadingen, Switzerland) in Eagle’s minimum essential medium (S-MEM, 0.15 % NaHCO3, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5 % fetal bovine serum (Gibco, Grand Island, NY, USA) at 37 °C in a humidified 5 % CO2 atmosphere.
Cells were fixed for 40 min at room temperature in 3 % paraformaldehyde and 0.1 % glutaraldehyde in Sörensen buffer (SB; 0.1 M Na/K phosphate buffer, pH 7.3). Upon washing in SB (including 20 min of incubation in 20 mM glycine in SB), the cells were centrifuged into 1 % low-melting agarose type VII (Sigma) in SB. The agarose-embedded cell samples were quickly dehydrated in a pre-cooled ethanol series (30, 50, 70, 96, and 100 %) and embedded in LR White resin (Polysciences Inc., Warrington, PA, USA) with polymerization under UV light for 48 h at 4 °C.
Cells were fixed for 40 min at room temperature in 3 % paraformaldehyde and 0.1 % glutaraldehyde in Sörensen buffer (SB; 0.1 M Na/K phosphate buffer, pH 7.3). Upon washing in SB (including 20 min of incubation in 20 mM glycine in SB), the cells were centrifuged into 1 % low-melting agarose type VII (Sigma) in SB. The agarose-embedded cell samples were quickly dehydrated in a pre-cooled ethanol series (30, 50, 70, 96, and 100 %) and embedded in LR White resin (Polysciences Inc., Warrington, PA, USA) with polymerization under UV light for 48 h at 4 °C.
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