We have previously described the production of MCTSs by seeding HNSCC cell lines into 384-well ULA-plates (cat. 4516; Corning, Tewksbury, MA).9 (link),20 (link),22 (link),25 (link) Briefly, 384-well ULA-plates were rehydrated by the addition of 50 μL of serum-free culture medium to each well and incubated in a humidified incubator for 15 min. Media was removed from the wells of the ULA-plates and 45 μL of a single-cell suspension of the HNSCC cell lines at a seeding density of 2500 cells/well in the appropriate growth medium was transferred into each well using a Matrix automated multi-channel pipette (Thermo Fisher Scientific). ULA-plates were centrifuged at 17g for 1 min and then placed in an incubator at 37 °C, 5% CO2, and 95% humidity for the indicated time periods. If HNSCC MCTS cultures were maintained in the ULA-plates beyond 3 days, spent media was exchanged for fresh medium every 3 days using a Janus MDT Mini (PerkinElmer, Waltham, MA) automated liquid handler platform equipped with a 384-well transfer head. Each medium exchange cycle consisted of 2 × 20 μL aspiration and discard steps, followed by 2 × 20 μL fresh media dispense steps. Three media exchange cycles were performed to achieve ~85% exchange of fresh medium for spent medium and a uniform volume of 45 μL/well.
Janus mdt mini
Manufactured by PerkinElmer
The Janus MDT Mini is a compact, automated liquid handling workstation designed for a variety of liquid handling applications in the laboratory. It features a 3-axis robotic arm and a modular deck to accommodate a range of labware and accessories, enabling automated pipetting, sample preparation, and other liquid handling tasks.
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2 protocols using janus mdt mini
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Generation of HNSCC Multicellular Tumor Spheroids
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Multicellular Tumor Spheroid Formation
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MCTSs were produced by seeding HNSCC cell lines into 384-well Ubottomed ultra-low attachment (ULA-plates) microtiter plates (Corning, Tewksbury, MA) as described previously. [ 25 , 36 , 38 , 49 , 52 ] Briefly, 384well ULA-plates were rehydrated by adding 50 μL of serum free medium (SFM) to wells and incubating for 15 minutes in a humidified incubator at 37°C before the media was removed and 45 𝜇L of a single-cell suspension of HNSCC cell lines, seeded at 2,500 or 5,000 cells/well in growth medium, were transferred into wells using a Matrix automated multichannel pipette (Thermo Fisher Scientific, Waltham, MA), ULA-plates were centrifuged at 17 x g for 1 minute, and then placed in an incubator at 37°C, 5% CO 2 and 95% humidity for the indicated time periods. For HNSCC MCTS cultures maintained in ULA-plates beyond 3 days, spent media was exchanged for fresh medium every 3 days using a Janus MDT Mini (PerkinElmer, Waltham, MA) automated liquid handler platform equipped with a 384-well transfer head. Each medium exchange cycle consisted of 2 × 20 μL aspiration and discard steps followed by 2 × 20 μL fresh media dispense steps. Three media exchange cycles achieved ∼ 85% media exchange and a uniform volume of 45uL per well.
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