For EB formation, human iPSC#16 cells were harvested after Accutase detachment and counted by Trypan Blue solution. Around 106 live cells were plated in each well of AggreWell 800 plate (Stem Cell Technologies) in AggreWell medium (Stem Cell Technologies) plus 10 μM ROCK inhibitor (Sigma). Forty-eight hours after plating, EBs were transferred into Ultra Low-attach six well-plate (Corning, USA) and the medium was replaced every other day. After 8 days as floating culture, EBs were transferred onto gelatin-coated plate and cultured in the same medium for another 8 days. On day sixteen the plates were fixed with 4% PFA solution and processed by immunostaining.
Aggrewell 800 plate
Manufactured by STEMCELL
Sourced in United States, Belgium, Canada
The AggreWell 800 plates are a laboratory product designed to facilitate the formation of uniform, 3D aggregates of cells. These plates feature an array of microwells that allow for the controlled aggregation of cells, enabling the production of consistent-sized spheroids or organoids. The core function of the AggreWell 800 plates is to provide a reliable and standardized platform for the generation of 3D cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (6 mentions)
Accutase, by STEMCELL (5 mentions)
Aggrewell medium, by STEMCELL (4 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Neurobasal a, by Thermo Fisher Scientific (3 mentions)
Sb431542, by Bio-Techne (3 mentions)
Neural induction medium, by STEMCELL (3 mentions)
N2 supplement, by Thermo Fisher Scientific (3 mentions)
Plo l, by Merck Group (2 mentions)
Stemdiff neural induction medium, by STEMCELL (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (2 mentions)
Rosette selection reagent, by STEMCELL (2 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Eb formation medium, by STEMCELL (2 mentions)
L ascorbic acid, by Merck Group (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
38 protocols using aggrewell 800 plate
1
Differentiation of iPSCs into EBs
2
Feeder-free iPSC Differentiation to Neural Progenitors
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Feeder-free iPSCs were dissociated with TryplE and seeded onto AggreWell™800 plate (10,000 cells per EB; Stem Cell Technologies) in E8 media supplemented with 10 μM ROCK inhibitor Y27632 for the first 24 h; we changed 75% of the media daily with STEMdiff™ Neural Induction Medium (NIM, Stem Cell Technologies). EBs were harvested after 5 days and plated onto poly-L-ornithine/laminin (PLO/L, Sigma) coated plates. 1–2 day(s) after attachment, prominent neural rosette structures were visible inside the attached neural aggregates. Rosettes were formed by cells expressing proteins characteristic of progenitor marker of PAX6, as described in Supplementary Information .
3
Directed Differentiation of iPSCs and hESCs
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iPSC and hESC colonies cultured for 24 h in Essential 8 medium supplemented with 10 μM of Y-27632 were dissociated to a single cell suspension by StemPro Accutase (Life Technologies) and counted. Embryoid bodies (EBs) composed of approximately 5000 cells were formed by using AggreWell 800 plate (Stem Cell Technologies) by seeding 1.5×106 cells per well. After 2 days, EBs were transferred to low-adhesion non-coated polystyrene plates (Nunc, part of Thermo Scientific, Waltham, MA, USA) and maintained in suspension culture in Essential 6 medium for 7–10 days. For endoderm differentiation, the EBs were transferred into RPMI medium supplemented with 2 % FCS, 100 ng/ml of Activin A, and 3 μM of CHIR99021 for 48 h. Mesoderm differentiation was initiated with DMEM/F12 medium supplemented with 10 % FCS, 500 μM L-ascorbic acid, and 100 μM β-mercaptoethanol. For ectoderm induction, the EBs were cultured in DMEM/F12 medium with N2/B27 supplements, 100 μM β-mercaptoethanol, and 1 × NEAAs. After the precondition step, the EBs were transferred onto poly-L-lysine-coated glass coverslips and cultured for 7 days in Essential 6 medium, fixed, and analysed for expression of the three embryonic germline layers markers by immunofluorescence.
4
Pluripotency Confirmation via Trilineage Differentiation
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Three germs layer differentiation was used to confirm the pluripotency of iPSCs using the STEMdiff Trilineage Differentiation kit (Stemcell Technologies) according to manufacturer’s instructions with some modification59 (link). When cells were ~70% confluent, iPSCs were passaged using Accutase (Stemcell Technologies) and plated into an AggreWell™800 plate (Stemcell Technologies) to form embryonic bodies (EB) in mTeSR1 medium for 2 days. EBs were washed out from the AggreWell plate and transferred to 6-well non-tissue culture treated plates (Corning) in specific differentiation mediums for each lineage. EBs were subjected to differentiation into mesoderm and endoderm lineages for 5 days or an ectoderm lineage for 7 days, then seeded onto Matrigel-coated plates for further analysis. Differentiation was assessed by immunostaining for germ layer-specific markers (Endoderm: SOX17; Mesoderm: Brachyury; Ectoderm: Nestin/Sox2).
5
Differentiation of Human iPSCs into 3D Cerebral Organoids
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Human iPSCs were differentiated into 3D cerebral organoids as previously described [33 (link), 41 (link)]. iPSCs were cultured as single cells in 10 µM of Y-27632 supplemented E8 media and transferred into individual wells of an AggreWell™800 plate (Stem Cell Technologies). The cells were incubated at 37 °C and 5% CO2 for 24 h. Organoids were then washed and transferred to an ultra-low attachment 10 cm plates in E6 medium supplemented with 2.5 μM Dorsomorphin (DM) (Tocris), 10 μM SB431542 (Tocris,) and 2.5 µM XAV-939 (Tocris). Media was replaced daily. On day 6 in suspension, media was replaced with neural medium (NM) containing Neurobasal-A (Life Technologies) supplemented with B27 supplement without vitamin A (Life Technologies), GlutaMax (Life Technologies), Anti-A (Life Technologies), 20 ng/ml FGF2 (R&D Systems) and 20 ng/ml EGF (Peprotech). Organoids were then cultured for an additional 19 days with daily medium changes in the first 10 days, and every other day for the final 9 days. To promote differentiation of the neural progenitors into neurons, FGF2 and EGF were replaced with 20 ng/ml BDNF (Peprotech) and 20 ng/ml NT3 (Peprotech) beginning at day 25. Beyond day 43, medium changes were performed every four days using only NM lacking growth factors.
6
Differentiation of iPSCs into Astrocytes
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iPSCs were maintained on Matrigel in StemFlex medium (Thermo Fisher Scientific) and passaged every 3–4 days with 0.5 mM EDTA dissociation solution. iPSCs were differentiated into neural progenitor cells (NPC) using an embryoid body (EB) protocol. Briefly, iPSCs at 80% confluence were collected, resuspended in Neural Induction Medium (NIM, StemCell Technologies) and seeded on one well of an Aggrewell 800 plate (StemCell Technologies) at 3 × 106 cells per well. At day five, EBs were seeded on poly-ornithine and laminin (PLO/LAM)-coated dishes in NIM. Rosette selection was performed after 12 days using Rosette Selection Reagent (StemCell Technologies). NPCs were expanded for 7 days in Neural Progenitor Medium (StemCell Technologies). NPCs were then differentiated into astrocyte precursors by seeding dissociated single cells at 1 × 105 cells/cm2 density on PLO/LAM dishes in STEMdiff astrocyte differentiation medium (StemCell Technologies). Astrocyte precursors were maintained for 20 days with medium changes every 48 hr and splitting every week with Accutase (Millipore, Burlington, MA). Astrocytes were expanded for up to 120 days in STEMdiff astrocyte maturation medium (StemCell Technologies).
7
Embryoid Body Formation from ESCs
8
Microglia-like Cells Generation from hiPSCs
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Microglia-like cells were generated following Haenseler et al. [19 (link)]. In brief, embryonic bodies (EBs) were formed through plating and spinning of 3X106 BIONi010-C (BINI-10) (WT, PLCG2HET and PLCG2HOM) at 300 g on an AggreWell 800 plate (34,850, StemCell Technologies) in E8-Flex medium supplemented with 50 ng/mL BMP4 (120-05ET, Peprotech), 50 ng/mL VEGF (PHC9394, ThermoFisher) and 20 ng/mL SCF (300-07, PeproTech). 75% medium change per day was performed for 72 h, after which EBs were transferred to a T75 flask and maintained in X-VIVO15 (BE02-060F, Lonza) supplemented with 25 ng/mL IL-3 (PHC0031, ThermoFisher), 100 ng/mL M-CSF (300–25, PeproTech), 2 mM Glutamax (35050061, ThermoFisher) and 0.055 mM β-mercaptoethanol (31350-010, ThermoFisher). Medium was topped up every week and after 4 weeks, emerging precursor cells were collected and differentiated to microglia-like cells for 7 days in microglia medium consisting of 25 mL DMEM/F12 (11330032, ThermoFisher) and 25 mL Neurobasal Plus media (A3582901, ThermoFisher) supplemented with 100 ng/mL M-CSF (300-25, Peprotech), 100 ng/mL IL-34 (200-34, peprotech) and 10 ng/mL GM-CSF (300-03, Peprotech), 2 mM Glutamax (35050061, ThermoFisher), 0.05 5 mM β-mercaptoethanol (31350, Life Technologies), and 0.25 μg/mL Insulin (I9278, Sigma).
9
Embryoid Body Formation from iPSCs
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Formation of embryoid bodies30 (link) was initiated with the dissociation of iPSCs into single cells using Accutase (STEMCELL Technologies). Approximately 1.5 × 106 cells/well were plated in an AggreWell 800™ plate (STEMCELL Technologies), which contained EB formation medium (STEMCELL Technologies) supplemented with 10 μM ROCK inhibitor (Y-27632, Tocris Bioscience) to form uniform embryoid bodies30 (link) overnight. The next day, embryoid bodies were transferred to petri dishes and refreshed with embryoid body formation medium every day. After 10 days of incubation, cells were fixed with 4% paraformaldehyde in PBS for immunofluorescence staining.
10
Generation of Brain Organoids from hiPSCs
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To generate brain organoids, hiPSC cultures were dissociated into a single cell suspension using Accutase (Innovative Cell Technologies, Inc., #AT-104) for 20 minutes at 37 °C, diluted with PBS, transferred to a 15 mL conical tube, and then centrifuged for 5 minutes at 300 x g. The supernatant was removed and the hiPSCs were resuspended in warm mTeSR Plus with 10 μM Rock inhibitor (StemCell Technologies #72304) and counted on an automated cell counter (Bio-Rad, #1450102). Approximately 3 × 106 hiPSCs were seeded per well of a 24-well AggreWell-800 plate (StemCell Technologies, #34815) which was centrifuged at 100 x g for 3 minutes to form spheroid aggregates (Day 0). After 24–48 hours, the spheroids were dislodged from the microwells and transferred to an Ultra-Low Attachment 6-well plate (Costar, #3471) using a wide bore P1000 pipette tip. The spheroids were maintained in suspension culture on an orbital shaker (~95 rpm) and guided via directed differentiation into brain organoids using previously described methods (S2 Table ) [46 (link)].
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