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.
Phc9394
Manufactured by Thermo Fisher Scientific
The PHC9394 is a laboratory incubator designed for precise temperature control and monitoring. It features a digital display for temperature readout and user-friendly controls for setting the desired temperature. The incubator is suitable for a variety of applications that require a stable and consistent temperature environment.
Automatically generated - may contain errors
Lab products found in correlation
Mini shaker, by Avantor (3 mentions)
Pmg0034, by Thermo Fisher Scientific (3 mentions)
24 well ultra low attachment plate, by Corning (3 mentions)
Clear round bottom ultra low attachment microplate, by Corning (2 mentions)
L ascorbic acid phosphate, by Fujifilm (2 mentions)
Phc3015, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide lps solution, by Thermo Fisher Scientific (1 mentions)
Recombinant human tgf β1, by R&D Systems (1 mentions)
Round bottom ultra low attachment microplate, by Corning (1 mentions)
4 protocols using phc9394
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
Cell Line Treatment Protocol
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Cell lines were treated with the following where relevant prior to downstream assays: 10 µg mL−1 TGF‐β Receptor 1 small molecule inhibitor (LY 364 947, Tocris), 5 ng mL−1 recombinant human TGF‐β1 (240‐B‐002, R&D Systems), 10 ng mL−1 Lipopolysaccharide (LPS) solution (00‐4976‐93, Invitrogen), 10 ng mL−1 recombinant human TNF‐α (PHC3015, Gibco), 10 ng mL−1 recombinant human IL‐6 (PHC0064, Gibco), 10 ng mL−1 recombinant human IL‐10 (PHC0104, Gibco), 10 ng mL−1 recombinant human VEGF‐A (PHC9394, Gibco), 1 ng mL−1 TNF‐α Receptor small molecule inhibitor (CAS 1049741‐03‐8, Calbiochem, Sigma–Aldrich).
4
Gastruloid Generation from mESCs
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Gastruloids were generated as previously described (Baillie-Johnson et al., 2015; (link)Rossi et al., 2020; (link)van den Brink et al., 2014) (link). Briefly, 300-700 mESCs were aggregated in 40μl N2B27 in 96-well Clear Round Bottom Ultra-Low Attachment Microplates (7007, Corning). After 48h, 150μl per well of 3μM Chi in N2B27 were added. At 72h, 150μl of medium were removed and substituted with 150μl of fresh N2B27. From 96h, the medium was changed to N2B27+++, which contains 30ng ml -1 bFGF (PMG0034, Gibco), 5ng ml -1 VEGF 165 (PHC9394, Gibco) and 0.5mM L-ascorbic acid phosphate (013-12061, Wako). From 120h on, half of the medium was changed daily. From 144h, N2B27 was used for daily medium changes. For immunofluorescence analysis, gastruloids at 96h were transferred in Ultra-Low Attachment 24-Well Plates (3473, Corning) with 100μl of medium, plus 700μl of fresh N2B27+++, and cultured on an orbital shaker placed at 37°C, 5% CO2 at 100rpm (VWR mini shaker), with the same culture schedule.
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