RCC cells (1 × 103) were suspended in 0.33% Bacto‐agar (Sigma‐Aldrich) and then layered over 0.5% Bacto‐agar in six‐well plates. On day 30, we counted the colonies after fixing them with methanol and staining them with Giemsa.
Bacto agar
Manufactured by Merck Group
Sourced in United States, China, Israel
Bacto agar is a solidifying agent derived from red algae, commonly used in microbiological and cell culture applications. It provides a firm, gel-like medium for the growth and isolation of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (4 mentions)
D glucose, by Merck Group (2 mentions)
Whatman, by Cytiva (2 mentions)
24 well plate, by Corning (2 mentions)
Bacto peptone, by Merck Group (2 mentions)
Bacto agar, by Thermo Fisher Scientific (2 mentions)
Methanol, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Gelred, by Biotium (2 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Lb broth, by Merck Group (2 mentions)
C300 image, by Azure Biosystems (2 mentions)
Neb 10 beta competent e coli high efficiency, by New England Biolabs (2 mentions)
Gibson assembly, by New England Biolabs (2 mentions)
Flat bottom 24 well plate, by Corning (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Ammonium sulfate, by Merck Group (1 mentions)
Invsc1, by Thermo Fisher Scientific (1 mentions)
D xylose, by Merck Group (1 mentions)
Alkali cation yeast transformation kit, by MP Biomedicals (1 mentions)
37 protocols using bacto agar
1
RCC Cells Colony Formation Assay
2
Anchorage-independent Growth Assay
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Anchorage-independent growth assays were performed in triplicate in 35 mm well plates. 72 hours after infection, cells were trypsinised in order to achieve a single-cell suspension and diluted at the same concentrations. Equal amounts of cells were then resuspended in complete DMEM supplemented with 0.3% of Bacto Agar (Sigma) and seeded on the top of a thick layer of DMEM supplemented with 0.6% Bacto Agar. For SW620 and HCT116, 5000 cells were seeded and 10000 for SW480. After 2–3 weeks, colonies were quantified by taking photographs of 10 random fields. For crystal violet staining dishes were fixed with methanol, stained for 3 hours with 0.01% crystal violet in PBS 40% methanol and de-stained with PBS overnight.
3
Soft Agar Colony Formation Assay
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H1299 (500 cells per 6-well) or A549 (1000 cells per 6-well) cells were suspended in 0.33% Bacto agar (Sigma-Aldrich) were layered over 0.5% Bacto agar. After incubation for 12 or 30 days, the cells were fixed and stained with Giemsa stain for calculating the number of colonies.
4
Soft Agar Assay for Cell Colonies
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Soft agar was carried out by growing 2, 000 cells in 0.4% bactoagar containing 10% FBS on a bottom layer of solidified 0.8% bactoagar (Sigma, Shanghai, China) with 10% FBS in a 24-well flat-bottom plate (Corning) as our previously described [26 (link)]. After incubation for 2–3 weeks at 37 °C in a humidified incubator, the colonies (containing ≥50 cells) were counted with an inverted optical microscope.
5
Yeast Transformation Using Alkali-Cation Kit
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The S. cerevisiae yeast strain, INVSc1 (genotype: MATa his3Δ1 leu2 trp1-289 ura3-52/MATα his3Δ1 leu2 trp1-289 ura3-52), was obtained from Invitrogen. Transformation of the INVSc1 yeast was performed using an Alkali-Cation Yeast Transformation kit (MP Biomedicals) according to the manufacturer's instructions with 2 mL yeast preculture grown overnight in 100 mL YPD medium to an absorbance at 660 nm equivalent to an optical density of 1.6 (DU-640 Spectrometer; Beckman Coulter, Fullerton, CA). Transformants were selected by plating onto 2% glucose complete minimal (CM) medium minus uracil plates (1.4 g yeast synthetic dropout medium supplement without histidine, tryptophan, leucine, or uracil [Sigma-Aldrich]; 6.7 g yeast nitrogen base with ammonium sulfate [Sigma-Aldrich]; 20 g D-glucose [Fisher Scientific, Fair Lawn, NJ]; 76 mg each of histidine, tryptophan, and leucine; and 20 g Bacto Agar [Sigma-Aldrich] in a final volume of 1 L Milli-Q water) or onto 2% xylose CM medium plates (1.4 g yeast synthetic dropout medium supplement without histidine, tryptophan, leucine, or uracil; 6.7 g yeast nitrogen base with ammonium sulfate; 20 g D-xylose; 76 mg each of histidine, tryptophan, leucine, and uracil; and 20 g Bacto Agar in a final volume of 1 L Milli-Q water).
6
Arabidopsis Seed Germination and Growth
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Arabidopsis seeds of Columbia (Col-0) ecotype and homozygous T3 mutant seeds were surface sterilized (10% bleach and 0.1% triton-X100 solution) before sowing on 0.5x MS agar plates (MS; pH 5.9, 0.8% Bacto agar, Merck) or modified germination medium (1 mM KNO3, 0.8% Bacto agar). Seeds were stratified for 2 days at 4°C, then transferred to an IPS750 incubator (Memmert, Schwabach, Germany) at ∼22°C for 2 weeks under long day conditions (16 h day and 8 h night). Seedlings were transplanted in peat moss bags (JiffyTM Products International AS, Kristiansand, Norway) and grown in controlled growth rooms under 10 h of light per 24 h at ∼22°C and 60% relative humidity with optimal fertilization of 7 mM nitrate.
7
Whole Embryo Explant Culturing
8
Mating and Sporulation Quantification
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Cells of opposite mating types were mated at 26 °C on sporulation agar (SPA) (1% d -glucose, 0.1% KH2PO4, 0.1% of 1000× vitamin (4.2 mM pantothenic acid, 81.2 mM nicotinic acid, 55.5 mM inositol, 40.8 mM biotin), and 0.0045% of each of adenine, histidine, leucine, uracil, lysine hydrochloride, pH 5.5 in 2% Bacto agar) (Sigma-Aldrich, St Louis, MO, USA). Cells were taken from the mating mixture at regular time points over 4 days to quantify the proportion of cells (n > 2000) that had undergone sporulation. Counting was performed using an Olympus CX31 light microscope (Olympus Corporation, Tokyo, Japan). The number of asci containing four spores over the total number of cells counted was expressed as % in Figure 3 and Figure 4 . Ascus containing less than four spores were scored as abnormal.
9
Bacterial Culture and Standardization
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Bacteria were cultured on tryptic soy agar (TSA), composed of tryptic soy broth (Oxoid, Basingstoke, United Kingdom) and 1g/L of bacto-agar (Sigma Aldrich, St. Louis, MO, USA), supplemented with 5% defibrinated horse blood (Oxoid, Basingstoke, UK). Bacterial cultures were incubated at 36 ± 1 °C for 72 h in anaerobic conditions (AnaeroGen™, Thermo Fisher Scientific, Waltham, MA, USA) in a jar to create the ideal growth conditions (CO2: 9–13%; O2 < 0.01%).
Bacterial strains were then subcultured in liquid medium using TSB with 5% defibrinated horse blood (Oxoid, Basingstoke, United Kingdom). The bacterial concentration was measured using the DensiCHECK densitometer (bioMérieux, Marcy l’Étoile, France) after 1 mL of culture centrifugation at 4000× g for 5 min and pellet resuspension in 1 mL of physiological solution. The resulting McFarland value was used to calculate the bacterial concentration CFU/mL (colony forming units/mL) for the following standardization at a ratio of 1:50 with SiHa cells.
Bacterial strains were then subcultured in liquid medium using TSB with 5% defibrinated horse blood (Oxoid, Basingstoke, United Kingdom). The bacterial concentration was measured using the DensiCHECK densitometer (bioMérieux, Marcy l’Étoile, France) after 1 mL of culture centrifugation at 4000× g for 5 min and pellet resuspension in 1 mL of physiological solution. The resulting McFarland value was used to calculate the bacterial concentration CFU/mL (colony forming units/mL) for the following standardization at a ratio of 1:50 with SiHa cells.
10
Anchorage-Independent Colony Formation
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An anchorage-independent colony formation assay was performed using soft agar. Briefly, 0.7% Bacto agar (Sigma Aldrich, Saint Louis, MO, USA) was plated and solidified as a base layer. Then, 0.3% Bacto agar containing 5000 cells and drugs were plated onto the base layer. The medium was replenished twice a week. After 10 to 14 days, colonies were stained with crystal violet (Sigma Aldrich) and counted under a microscope (Zeiss, Berlin, Germany).
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