The cell colony forming assay was performed as previously described (20 (link)). Briefly, basal agarose was prepared with 1X DMEM, 0.6% low-melting-point agarose (Amresco, LLC), 10% FBS, 1% penicillin and 1% streptomycin. The basal agarose contained 0.2, 0.4 or 0.8 mM NGR1. The basal agarose compounds were poured into a 6-well plate. The top agarose layer was prepared with 1X DMEM, 0.3% low-melting-point agarose, 10% FBS, 1% penicillin and 1% streptomycin. Subsequently, cells (1×103 cells/well) were added to make the top agarose. The top agarose also contained 0.2, 0.4 or 0.8 mM NGR1. The top agarose compounds were poured onto the basal agarose. Cells were incubated at 37°C for 12 days. Subsequently, cell colonies were fixed with 4% paraformaldehyde for 20 min at room temperature, and stained with 0.5% crystal violet for 15 min at room temperature. Data were analyzed using ImageJ software (version 1.8.0; National Institutes of Health).
Low melting point agarose
Manufactured by Avantor
Sourced in United States
Low-melting-point agarose is a laboratory reagent used for the preparation of gels in various biological applications. It is characterized by a lower melting point compared to standard agarose, allowing for gentler handling and manipulation of samples during gel formation and analysis.
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16 protocols using low melting point agarose
1
Cell Colony Forming Assay with NGR1
2
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.
3
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.
4
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).
5
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 ].
6
Anchorage-Independent Cell Growth Assay
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Anchorage-independent growth was determined by a soft agar assay as described previously.24 (link) Briefly, a bottom layer of 1.4 mL (0.6%) was prepared with a 1:1 mixture of 1.2% low melting-point agarose (AMRESCO, OH, USA) (approximately 50°C) and warm 2 × DMEM, and was poured into each well of six-well plates. A top layer, which was a mixture of 500 μL of warm 2 × DMEM, 500 μL of the 0.6% base agar and cells at the density of 1 × 103 cells per well, was poured on top of the solidified bottom layer. The cultures were fed 1 mL of DMEM supplemented with 10% FBS, which was gently refreshed twice a week. The plates were kept in a cell culture incubator maintained at 37°C under 5% CO2 to allow colony growth. After 2 weeks of culture, the colony assay was terminated and cell colonies of more than 50 cells in a cluster were counted using a graduated eyepiece fitted in a transmission light microscope at a magnification of 40.25 (link)
7
Viral Plaque Assay Protocol
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Serial 10-fold dilutions of each viral sample were prepared in DMEM (plus 1% FBS) and inoculated onto BHK-21 monolayers in 6-well plates. The plates were incubated at 37 °C for 1 h. After inoculum removal, the cells were washed twice with PBS and overlaid with DMEM containing 1% (wt/vol) low-melting point agarose (Amresco, Houston, TX, USA) and 2% FBS. After further incubation at 37 °C for 72 h, the cells were stained with 0.02% neutral red to visualize the plaques.
8
Soft Agar Colony Formation Assay
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The assay was first performed in L-15 containing 0.6% low melting point agarose and 10% FBS. We seeded 1000 cells in 1 ml of L-15 containing 10% FBS and 0.3% low melting point agarose (Amresco, Cleveland, OH, USA) and laminated it to the substrate. Two weeks later, the plates were stained with 0.2% crystal violet and the number of cell colonies was counted under an optical microscope (IX70; Olympus Corporation, Tokyo, Japan) [19 (link),20 (link)].
9
Calcium Imaging of Arabidopsis Root Responses
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After germination, the Arabidopsis seedlings were grown vertically on the half-strength MS medium for 5–7 days and their roots were prepared for Ca2+ imaging following Krebs et al. (2012) (link), with some modifications. The roots were immobilized by overlaying them with 1% (w/v) low-melting-point agarose (Amresco) in the Attofluor®Cell Chambers (Invitrogen). After digging a small tunnel in the agarose to expose the root, we gently applied 200 μl bathing solution buffer [0.5x MS salt, 1% sucrose, 10 mM 2-(N-morpholino)ethanesulfonic acid [MES]-KOH, pH 5.8] to the chamber. The shoot was not submerged in the solution. High concentrations of NaCl and sorbitol in the same solution were separately perfused as the stimulus into the chamber. The mature zone of the Arabidopsis roots was selected for the subsequent Ca2+ measurements.
10
Clonogenic Assay for Cell Viability
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For clonogenic assay, the cells were treated with indicated concentrations of SNG1153 or vehicle (DMSO) respectively. After 24 h, the treated cells were suspended in 1 ml medium containing 0.3% low-melting-point agarose (Amresco, Solon, OH, USA), and plated on a bottom layer containing 0.6% agarose in 35 mm plates (1000 cells/plate). After 10 days of culture, cells were stained with Giemsa and colonies containing more than 50 cells were counted and photographed.
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