MCF-7 or MDA-MB-231 cells (~2,500) were resuspended in 0.35% SeaPlaque agarose (Lonza, 50101) in complete medium containing 0 nM, 17.8 nM, or 178 nM Cerberus-Fc and plated on a layer of 0.7% SeaPlaque agarose containing complete medium. Cells were fed twice weekly with complete medium and the corresponding concentrations of Cerberus-Fc. After three weeks, cells were stained with 0.005% crystal violet and images were taken. Cell clusters were quantified using ImageJ software. Colony forming ability was calculated by dividing the number of colonies by the number of initial cells. Percent colony forming ability was determined by dividing the number of colonies formed with Cerberus-Fc relative to no Cerberus-Fc control.
Seaplaque agarose
Manufactured by Lonza
Sourced in United States, Switzerland, Japan
SeaPlaque agarose is a low-melting temperature agarose used for the preparation of soft agar overlays for plaque assays. It has a gelling temperature of approximately 30-35°C and a melting temperature of approximately 60-65°C.
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Lab products found in correlation
Neutral red, by Merck Group (23 mentions)
Penicillin streptomycin, by Lonza (12 mentions)
Crystal violet, by Merck Group (10 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (6 mentions)
Neutral red solution, by Merck Group (5 mentions)
Tpck treated trypsin, by Merck Group (3 mentions)
Agarose, by Lonza (3 mentions)
Gelcount, by Oxford Optronix (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Vt1000, by Leica (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Minimum essential medium (mem), by Lonza (2 mentions)
Renilla luciferase assay kit, by Promega (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
188 protocols using seaplaque agarose
1
Cerberus-Fc Inhibits Breast Cancer Colony Formation
2
Colony Formation Assays for 2D and 3D Cell Culture
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For two-dimensional, anchorage-dependent colony formation assays, cells in single-cell suspension were seeded in triplicate wells of 6-well plates, treated with the designated compound(s), and allowed to form colonies for 7-14 days, depending on the cell line. Following incubation, cells were xed in methanol, stained with crystal violet, washed with water, and air-dried overnight. Pictures of the wells were captured and colonies were counted using Count and Plot Histograms of Colony Size (countPHICS) software 34 (link) , a macro written for ImageJ. The threshold for colony size was automatically determined by countPHICS.
For three-dimensional, anchorage-independent (soft agar) colony formation assays, cells in single-cell suspension were seeded into a 0.4% SeaPlaque ™ Agarose (Lonza Bioscience) liquid solution and placed on top of a solidi ed layer of 0.8% SeaPlaque ™ Agarose solution in triplicate wells of 6-well plates. The top layer with cells was allowed to solidify at room temperature for at least 30 minutes. Cells were then treated with the designated compound(s) and allowed to form colonies for 14 days. Following incubation, cells were stained with crystal violet and washed with water. Pictures of the wells were taken, and colonies were counted using countPHICS 34 (link) . The threshold for colony size was automatically determined by countPHICS.
For three-dimensional, anchorage-independent (soft agar) colony formation assays, cells in single-cell suspension were seeded into a 0.4% SeaPlaque ™ Agarose (Lonza Bioscience) liquid solution and placed on top of a solidi ed layer of 0.8% SeaPlaque ™ Agarose solution in triplicate wells of 6-well plates. The top layer with cells was allowed to solidify at room temperature for at least 30 minutes. Cells were then treated with the designated compound(s) and allowed to form colonies for 14 days. Following incubation, cells were stained with crystal violet and washed with water. Pictures of the wells were taken, and colonies were counted using countPHICS 34 (link) . The threshold for colony size was automatically determined by countPHICS.
3
Soft-agar Colony Formation Assay
4
Cellular Proliferation Assays for p53
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For cell accumulation assays, p53ERTAMKi/− MEFs were plated in 6-well plates at a density of 200,000 cells per well, transduced with RasV12 retrovirus as described above, and treated with 4OHT to restore p53. 4OHT treatment then was continued or withdrawn depending on the condition, and 7 days later cells were fixed with 3:1 methanol-acetic acid and stained with 0.5% cresyl violet in methanol. For the soft-agar colony formation assay, 0.8% base layer SeaPlaque agarose (Lonza) in 1.5 ml DMEM containing 10% fetal bovine serum, 1% nonessential amino acids, 1% sodium pyruvate, 1% glutamine, and 1% Pen-Strep was plated onto each well of 6-well plates, and cells suspended in 0.48% SeaPlaque agarose (Lonza) were added to the agar. Medium was replaced every 3 days. The plates were incubated at 37°C in 5% CO2, and cells were treated as described above for the accumulation assay. Two weeks later the clones were counted and photographed.
5
Anchorage-Independent Cell Growth Assay
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Twelve-well plates were coated with 0.8% agar (low melting SeaPlaque Agarose, Lonza) in DMEM supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) and 1× penicillin/streptomycin (Gibco, Thermo Fisher Scientific). On top, 1,500 cells per well in 0.4% agar (low melting SeaPlaque Agarose, Lonza) in DMEM with 10% FBS and 1× penicillin/streptomycin were seeded and air dried for 45 minutes at room temperature. After incubation at 37°C and 5% CO2 for 24 hours, 500 μL medium was added. Medium was changed twice a week and after 14–16 days, colonies were stained with 500 μL 0.005% Crystal Violet (Sigma, Merck) for 1 hour. Then destaining by washing three times with PBS was performed and pictures of colonies were taken on a AZ100 Zoom Microscope (Nikon).
6
Preparation of 0.4% SeaPlaque Agarose Solution
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For the preparation of 100 mL of 0.4% SeaPlaqueTM agarose (Lonza, Basel, Switzerland), 0.8 g of SeaPlaqueTM agarose powder was added to an Erlenmeyer glass flask containing 100 mL of Ampuwa water. The mixture was boiled until the agarose was completely dissolved. The solution was further diluted 1:1 in serum-free medium [21 (link)] to prepare a 0.4% agarose solution.
7
Anchorage-independent Growth Assay
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Anchorage-independent growth of osteosarcoma cells in the presence of TIIA was assayed by colony formation in soft agar. Cells were seeded 2 × 104 cells/well in six-well plates containing 0.5% SeaPlaqueTM agarose (Lonza, Rockland, ME, USA) media. After 3 weeks, the numbers of colonies were counted at least 100 mm in the diameter.
8
SARS-CoV-2 Plaque Assay in Vero Cells
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Vero cells were purchased from the Korean Cell Line Bank and cultured in Dulbecco’s minimum essential medium (DMEM) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 5% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. For plaque assay, Vero cells were seeded in a 6-well plate 1 day before the assay. Following this, cells were infected with SARS-CoV-2 serially diluted in serum-free medium for 1 h with gentle agitation every 15 min. The cells were then overlaid with DMEM containing 1% SeaPlaqueTM agarose (Lonza, Switzerland), 2% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. After 3 days of incubation until observation of clear plaques, 4% paraformaldehyde was used for fixation, and a 0.5% crystal violet-20% methanol solution was used for staining the cells. The number of plaques observed was multiplied by the dilution factor to calculate the virus titer.
9
3D Soft Agar Colony Assay
10
Agarose-Embedded Cell Differentiation
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Agarose gels were formed by dissolving SeaPlaqueTM Agarose (Lonza, Rockland, ME USA) in serum-free culture medium to a concentration of 2 wt.% at temperatures above 70 °C. The agarose solution was held at 37 °C in a water bath during cell preparation. An equal volume of warm cell suspension (20 × 106 cells/mL) and 2% agarose were mixed to achieve a final cell concentration of 10 × 106 cells/mL and a final agarose concentration of 1%. A volume of 75 µL of this mixture was immediately pipetted onto each ~15 mg dextran-fabric contained within separate wells of a 6-well culture plate. After 5 minutes of gelation, 2 mL of growth medium was added to each well. The following day, the growth medium was replaced with differentiation medium and the cells were cultured for an additional 6 days. The differentiation medium was replenished on days 3 and 5. On day 7, the samples were either lysed in NP-40 buffer containing SIGMAFAST protease inhibitor for Western blot analysis or fixed in 3.7% formalin for 30 minutes at room temperature for subsequent immunolabeling and staining. A minimum of 3 independent experiments were analyzed by immunolabeling and Western blot.
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