Gastruloids were generated as previously described (Baillie-Johnson et al., 2015 ). Briefly, 300–700 mESCs were plated in 40 μL N2B27 in 96-well Clear Round Bottom Ultra-Low Attachment Microplates (7007, Corning). After 48 h, 150 μL of N2B27 containing 3 μM Chi were added to each well. After 72 h, medium was changed with N2B27. Starting from 96 h, the protocol was optimized as described in Figure S1A . At 96 h, gastruloids were transferred in Ultra-Low Attachment 24-well Plates (3473, Corning) in 100 μL of medium, plus 700 μL of fresh N2B27 containing 30ng ml−1 bFGF (PMG0034, GIBCO), 5ng ml−1 VEGF 165 (PHC9394, GIBCO) and 0.5mM L-ascorbic acid phosphate (013–12061, Wako) (N2B27+++) and cultured on an orbital shaker placed at 37°C, 5%CO2 at 100rpm (VWR mini shaker). From 120 h onward, half medium was changed daily. Unless differently specified, N2B27+++ was applied from 96 to 144 h, while from 144 h to 168 h N2B27 was used for medium change. To generate Hcn4-GFP::Tbx1Cre-RFP gastruloids, 800–1200 cells were employed, due to initial difficulties in cell aggregation and extreme susceptibility to Chi treatment. For the same reason, Chi pulse for this line was done with 1 μM Chi, with the exact same modalities described for the other lines.
Clear round bottom ultra low attachment microplate
Manufactured by Corning
Sourced in United States
The Clear Round Bottom Ultra Low Attachment Microplate is a laboratory equipment product designed for cell culture applications. It features a clear round bottom well design and a specialized surface treatment to minimize cell attachment, promoting the growth of suspension cultures.
Automatically generated - may contain errors
Lab products found in correlation
Mini shaker, by Avantor (2 mentions)
Pmg0034, by Thermo Fisher Scientific (2 mentions)
Phc9394, by Thermo Fisher Scientific (2 mentions)
24 well ultra low attachment plate, by Corning (2 mentions)
Evos m500, by Thermo Fisher Scientific (2 mentions)
L ascorbic acid phosphate, by Fujifilm (1 mentions)
Methionine free rpmi, by Thermo Fisher Scientific (1 mentions)
Te2000 u, by Nikon (1 mentions)
Axiocam 305 mono camera, by Zeiss (1 mentions)
Axio vert a1 inverted microscope, by Zeiss (1 mentions)
17 protocols using clear round bottom ultra low attachment microplate
1
Optimized Gastruloid Generation Protocol
2
Gastruloid Generation and Immunofluorescence Analysis
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Gastruloids were generated as previously described6 (link),9 (link),69 (link). Briefly, 300–700 mESCs were aggregated in 40 μl N2B27 in 96-well Clear Round Bottom Ultra-Low Attachment Microplates (7007, Corning). After 48 h, 150 μl per well of 3 μM Chi in N2B27 were added. At 72 h, 150 μl of medium were removed and substituted with 150 μl of fresh N2B27. From 96 h, the medium was changed to N2B27+++ which contains 30 ng ml−1 bFGF (PMG0034, Gibco), 5 ng ml−1 VEGF 165 (PHC9394, Gibco) and 0.5 mM L-ascorbic acid phosphate (013-12061, Wako). From 120 h on, half of the medium was changed daily. From 144 h, N2B27 was used for daily medium changes. For immunofluorescence analysis, gastruloids at 96 h were transferred in Ultra-Low Attachment 24-Well Plates (3473, Corning) with 100 μl of medium, plus 700 μl of fresh N2B27+++, and cultured on an orbital shaker placed at 37 °C, 5% CO2 at 100 rpm (VWR mini shaker), with the same culture schedule.
3
3D Spheroid Drug Response Assay
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3D multicellular spheroids were generated as previously described.24 Briefly, cells were seeded in 96‐well Clear Round Bottom Ultra‐Low Attachment Microplates (Corning, cat# 7007) and monitored daily. Seeding densities were optimised for H460 cells so that tumour spheroids fell within a size range of approximately 300–500 μm, and were of consistent macroscopic morphology (aspect ratio) across wells on day 4. Spheroids with an average diameter ~ 300–500 μM after 4 days growth were treated with either DMSO or 0–5 μM MP‐470 and imaging commenced. Images were captured every 2 h using the 4x objective (NA 0.2) of an IncuCyte S3 widefield inverted live cell system (Essen BioScience) housed inside a cell culture incubator (37°C, 5% CO2). Spheroid size (area) was measured using ImageJ software (NIH, V.1.53c) and calculated as fold‐change from time = 0 h to account for any initial differences in spheroid size.
4
Quantitative Analysis of Tumor Spheroid Growth
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2.5 × 103 HeLa cells were seeded in 96-well Clear Round Bottom Ultra-Low Attachment Microplates (7007, Corning®) in a final volume of 200 μl. Cells were centrifuged at 2,000 rpm for 10 min to encourage the formation of a single spheroid per well. 12 replicates of each condition were plated in each biological replicate. Every three days, 100 μl cell culture medium was replaced, and every two days spheroids were photographed in an EVOS™ M500 (Thermo Fisher Scientific™) at 4× magnification. Spheroids were cultured for 7 days. Fiji Image J Software was used to measure the volume and circularity of spheroids. Spheroid area was calculated as previously reported61 (link), and used to determine spheroid radius (R = √(area/π)), from which the volume (V = (4/3) πR3) was calculated. Each spheroid’s GFP levels were quantified using Fiji Image J Software and the intensity relative to spheroid volume was depicted.
5
Quantitative Analysis of Tumor Spheroid Growth
6
Selective Protein Labeling in Cells
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Selective labeling of actively synthesized proteins was performed as described by Dieterich et al. 2006. In brief, cells were lifted from plates and washed of complete media with 3x PBS washes prior to resuspension in methionine or HPG supplemented methioninefree RPMI (Thermo Scientific). Cells were then introduced to 96-well clear round bottom ultra-low attachment microplates (Corning) at 50,000 cells per 100µL and incubated 2hrs at 37°C. Cells were then washed 2x with PBS to remove excess methionine or HPG before pellets were snap frozen and stored at -80°C.
7
Tumor Sphere Formation Efficiency
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Cells were cultivated in 96-well Clear Round Bottom Ultra Low Attachment Microplate (Corning). After 7 days, the tumor spheres formed in the microplate were observed and counted. The percentage of cells which could develop into a tumor sphere was regarded as sphere formation efficiency.
8
Tumor Sphere Formation Assay
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Colorectal cancer cells were seeded onto commercial 96‐well Clear Round Bottom Ultra Low Attachment Microplate (Corning Incorporated). After incubation for 7 days, the number of tumor spheres was analyzed under a microscope. Sphere formation efficiency was analyzed as the proportion of cells which could form tumor spheres.
9
Sphere Formation Assay for NSCLC Cells
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NSCLC cells were seeded onto Clear Round Bottom Ultra Low Attachment Microplate (Corning) and incubated for 7 d. The diameter of spheres was assessed under a microscope. Sphere formation assay was conducted three times with three biological repetitions each time.
10
Renal Cell Differentiation of USCs
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