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Agarose

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Sourced in United States, China, Germany

Agarose is a polysaccharide derived from seaweed that is commonly used as a gelling agent in various laboratory applications. It is a versatile material that forms a porous, semi-solid matrix when dissolved in water and allowed to cool. Agarose is often used in gel electrophoresis techniques to separate and analyze macromolecules such as DNA, RNA, and proteins based on their size and charge.

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36 protocols using agarose

1

Cell Proliferation and Colony Formation Assays

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After different types of gene perturbations, the cells were cultured in 48-well plates, at the starting density of 20,000 cells per well. The IncuCyte live-cell imaging and analysis system (ESSEN Bioscience) was used to monitor real-time cell proliferation and morphology changes. Cell proliferation was quantified by measuring the occupied area (% confluence) in the cell images over time. The proliferation curves were made with confluence fold change (FC) at different time points in relative to the confluence at time 0.
For colony formation assays, 1000 cells were seeded in the 6-well plates and incubated with normal growth medium for 14 days. Then cells were fixed and stained with 0.5% crystal violet for 15 min. Lastly, the colonies were imaged via a camera or a microscope. For the anchorage-independent colony formation assay, the cells were collected, resuspended in culturing media containing 10% FBS and 0.3% agarose (Amresco), and then seeded on top of 0.6% agarose gel containing 10% FBS in six-well plates. The cells were cultured in regular media for 3–4 weeks, and the colonies were stained with crystal violet and photographed.
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Soft Agar Colony Formation Assay

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MCF7 and T47D cells stably expressing the shRNAs were collected, resuspended in culturing media containing 10% FBS and 0.3% agarose (Amresco), and then seeded on top of 0.6% agarose gel containing 10% FBS in six‐well plates. The starting point of cell density was 1,000 cells per well. The cells were cultured in regular media for 3–4 weeks, and the colonies were stained with 0.005% crystal violet and photographed.
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3

Soft Agar Colony Formation Assay

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Cells were suspended in 1 ml of RPMI 1640 containing 0.3% low-melting-point agarose (Amresco, Cleveland, OH, USA) and 10% FBS, and plated on a bottom layer containing 0.6% agarose and 10% FBS in a six-well plate in triplicate. After two weeks, plates were stained with 0.2% gentian violet and the colonies were counted under a light microscope (IX70; Olympus Corporation, Tokyo, Japan) after two weeks.
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4

Virus Plaque Assay in MDCK Cells

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MDCK monolayer cells in 12-well dishes were washed twice with PBS, followed by infection with serial 10-fold dilutions of virus in serum-free DMEM medium supplemented with 4 μg/ml of TPCK-trypsin (Sigma), and incubated at 37 °C for 1 h. Afterwards, the cells were washed with PBS and overlaid with Modified Eagle’s Medium (MEM) containing 1% agarose (AMRESCO) and 2 μg/ml of TPCK-trypsin. After 2 days, visible plaques were counted and viral titers were calculated.
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5

Viral Plaque Assay in MDCK Cells

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MDCK cells were seeded in 12-well plates and infected with serial dilutions of the virus in serum-free DMEM supplemented with 4 μg/mL of TPCK-trypsin for 2 h, and then washed with PBS. The cells were covered with Modified Eagle’s Medium containing 1% agarose (AMRESCO) and 2 μg/mL of TPCK-trypsin. The plates were allowed to solidify at 4 °C for 5 min and incubated upside-down at 37 °C. After 72 h, viral titers were determined through counting the visible plaques.
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6

Cell Colony Formation Assay

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Cell colony formation assay was performed as previously described [44 (link)]. Briefly, the treated cells at 1 × 103 cells/well were suspended in 1 mL of DMEM containing 0.6% low-melting-point agarose (Amresco, USA) and 10% FBS, and plated on a bottom layer containing 0.3% agarose and 10% FBS in 6-well plate in triplicate. The 6-well cell plates were added Ary at 1.25, 2.5, 5, 10 or 20 μg/ mL. After being incubated for two weeks, the cell plates were stained with 0.1% crystal violet for 15 min, and the colonies were counted under a microscopy.
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7

Soft Agar Colony Formation Assay

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A549 and H1975 cells (1000 cells per plate) were suspended in 1 mL of DMEM or RPMI 1640 containing 0.3% low-melting-point agarose (Amresco, USA), 10% FBS and indicated concentration of HHT, andplated on a bottom layer containing 0.6% agarose and 10% FBS in 6-well plate intriplicate. After 2 weeks, plates were stained with 0.5 mL of 0.005% crystal violet for more than 1 h and the colonies were counted under light microscope60 (link).
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8

Soft Agar Colony Formation Assay

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Cells were suspended in 1 ml of DMEM or L-15 containing 0.3% low-melting-point agarose (Amresco, USA) and 10% FBS, and plated on a bottom layer containing 0.6% agarose and 10% FBS in 6-well plate in triplicate. After 2 weeks, plates were stained with 0.2% gentian violet and the colonies were counted under light microscope [6 ].
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9

Reagents and Equipment for Nucleic Acid Analysis

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Reagents (of analytical purity) Chelex-100 and betaine were purchased from Sigma, USA; manganese chloride, magnesium sulfate, potassium chloride, sodium hydroxide, EDTA, and ammonium sulfate from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); Tris-HCl from the Shanghai Shengke Biotechnology Co., Ltd. (Shanghai, China); Triton X-100 from the Beijing Meilaibo (MyLab) Medical Technology Co., Ltd. (Beijing, China); dNTPs from Pharmacia, USA; NP-40 from Fluka, USA; 2× Tap MIX Kit from the Tiangen Biochemical Technology Co., Ltd. (Beijing, China); Agarose from Amresco, USA; LAMP DNA Amplification Kit, LAMP reaction tube, and calcein (the fluorescent detection reagent) from the Eiken Chemical Co., Ltd. (Tochigi, Japan); the real-time turbidity meter (LA-320C) from the Rongyan Chemical Co., Ltd. (Tokyo, Japan); thermostatic metal bath (HB-2) from the Wealtech Corporation (Sparks, NV, USA); Spectrophotometer (NanoQTM) from the CapitalBio Technology Co., Ltd. (Beijing, China); gel imaging system (Quantun -ST5) from the Gel imager VILBER (Beijing, China); and PCR thermal cycler (ETC811) from the Gene Amplifier Suzhou Dongsheng Xingye Scientific Instrument Co., Ltd. (Suzhou, China).
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10

cDNA Synthesis and Gel Electrophoresis

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For cDNA synthesis 1 μg of each RNA sample was reverse-transcribed. PCR products in agarose (Amresco, Solon, OH, USA) gels, visualized by ethidium bromide (EtBr, Sigma-Aldrich, St. Louis, MO, USA) staining were digitally photographed for intensity analysis.
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