Regionally specified organoids were initially differentiated as described above and induced to fuse together at 50 days of differentiation to generate assembloids following protocols established for other systems (Sloan et al., 2018 (link)). Briefly, to generate assembloids between retinal and prosencephalic brain organoids, a single POU4F2:tdTomato-positive retinal organoid was fused with a single brain organoid by placing each into a single 1.5 mL centrifuge tube and incubating at 37°C at 5% CO2 to allow the organoids to fuse together. Three days later, fused assembloids were transferred to a single well of a low-attachment 24-well plate (Corning) for further maturation in RDM supplemented with 10% FBS, 1× Glutamax, and 100 μM taurine, with the medium changed every 2–3 days. Tri-assembloids with retinal, thalamic, and cortical organoids were established upon membranes to allow for more controlled positioning of organoids. Three days after assembly, tri-assembloids were transferred into individual wells of a low-attachment 24-well plate and maintained as outlined above. At indicated time points, assembloids were collected and fixed in 4% paraformaldehyde before cryostat sectioning for immunocytochemical analyses.
24 well low attachment plate
Manufactured by Corning
Sourced in United States
The 24-well low-attachment plates are a laboratory equipment product designed for cell culture applications. These plates feature a surface treatment that prevents cells from adhering, allowing for the formation of spheroids, organoids, or suspension cultures. The core function of these plates is to facilitate the growth and maintenance of cells in a non-adherent environment.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Spectramax id3, by Molecular Devices (2 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Facsdiva software, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Soybean trypsin inhibitor, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 hydroxytamoxifen, by Merck Group (1 mentions)
Collagenase p, by Roche (1 mentions)
Cell death elisa, by Roche (1 mentions)
Alamar blue solution, by Merck Group (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
Vegf a, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Stereomicroscope, by Nikon (1 mentions)
Sb43152, by Selleck Chemicals (1 mentions)
Tib 71, by American Type Culture Collection (1 mentions)
34 protocols using 24 well low attachment plate
1
Assembling Multifaceted Brain Organoids
2
Assessing BM-MSCs Viability on SNC
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Both pooled older donor and young donor BM-MSCs (3 × 104 cells) were loaded separately onto preconditioned SNC samples (10 mm diameter × 1 mm height) in triplicate in 24-well plates and incubated for 1 h. After the incubation, the SNC samples were moved to a low-attachment 24-well plate (Corning, NY, USA) and cultured for 7 days in SM medium. On day 7, the viability of the BM-MSCs on the SNC samples was assessed. CellTrackerTM Green (Thermo Fisher Scientific, Waltham, MA, USA) was prepared in serum-free DMEM medium, to a final working concentration of 0.7 µM and the samples were stained for 30 min at 37 °C and 5% CO2. Subsequently, the samples were imaged using confocal microscopy (Leica TCS SP8 confocal microscope, Leica Microsystems, Wetzlar, Germany). The cells present in each image (n = 3) of the SNC loaded with young and older donor BM-MSCs were counted using Image J software v1.52 and the average number of cells in each scaffold were calculated and normalised to the surface area.
3
Neural Induction of hEBs
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hEBs that reached a diameter of ca. 500–650 μm after 6 days and had a smooth surface, were transferred into separate wells of a low attachment-24-well plate (Corning, Wiesbaden, Germany), filled with Neural Induction Medium. Incubation for 5 days at 37 °C and 5% CO2 followed. Medium was replaced after 48 h.
4
Lentiviral Transduction of Cryogel Scaffolds
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To immobilize the lentivirus on the scaffolds, cryogels (n = 4/group/timepoint) were incubated overnight with 0.002% poly-L-lysine (Sigma), washed with PBS, and then incubated with enough concentrated lentivirus to transduce 500,000 or 1 million cells for 1.5 h at 37°C. The scaffolds were rinsed again with PBS, moved to a new low-attachment 24-well plate (Corning), and seeded with 500,000 cells for in vitro experiments and 1 million cells for implanted scaffolds. The expansion medium was added to each sample for 1 h after the cell seeding. All mediums were changed 24 h later. The scaffolds were cultured in the expansion medium for six days to facilitate cell infiltration. A subset of scaffolds were then fixed in a neutral-buffered formalin (NBF) for 24 h, placed in a 30% sucrose solution for an additional 24 h, and embedded in an optimal cutting temperature (OCT) compound. Once frozen, the cryogels were cryosectioned at 20 µm and stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the cellular infiltration. All other scaffolds remained in culture.
5
Optimizing hiPS Cell Expansion on Microcarriers
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The protocol for the screening of microcarriers for hiPS cell expansion under static culture in low-attachment 24-well plates (Corning Inc.) was recently published [27 (link)]. We used 3 cm2 of microcarrier superficial area per well and cells were inoculated at an initial density of 5x104 cells/cm2. Geltrex- and vitronectin-coated polystyrene microcarriers (GM and VtnM) were tested. 80% of E8 medium was changed daily for 5 days. The cell yield in total cell number was calculated as the ratio Xday5/Xi, where Xday5 is the number of viable cells, attached to the microcarriers, at day 5, and Xi is the number of cells inoculated at day 0.
6
Macrophage Polarization and Poly(I:C) Uptake
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Human monocytes were purified and polarized toward M0 macrophages as described in section Human Primary Macrophage Differentiation. These cells were seeded at a density of 1 × 106 cells/well in low-attachment 24-well plates (Corning, ME, USA), and 0.5 mL of fresh RPMI media containing poly(I:C), either free or nanocomplexed, were added to them. The final poly(I:C) dose per well was of 5 μg/mL of poly(I:C), of which 0.25 μg/mL were poly(I:C)-rhodamine. After 24 h of incubation, cells were detached from the wells with trypsin-EDTA. Cells were then washed one time with FACS buffer (PBS 1% BSA) and fixed in FACS Fix (PBS 1% PFA) for 20 min at 4°C. Cell suspensions were centrifuged at 1,750 rpm for 10 min and 4°C. The supernatants were then discarded, and cells re-suspended in 300 μL of FACS buffer (PBS 1% BSA). Treated macrophages were analyzed by flow cytometry using a BD LSR FortessaTM (BD Biosciences, CA, USA), and the resulting data analyzed by FACS Diva software (BD Biosciences, CA, USA), determining the mean fluorescence intensity (MFI) of rhodamine-positive cells. Results were expressed as fold change in comparison to the free poly(I:C)-rhodamine.
7
Neurosphere Formation Assay for Glioma Cells
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U373, SNB19 and U87 transfected cells were plated at the maximum density of 4 000 cells/mL in low-attachment 24-well plates (Corning). Cells were cultured in serum-free stem cell medium, as detailed above. Neurospheres were supplemented with fresh media every 4-5 days (250 µL/well). The number of neurospheres was counted after 10 days for U373 and U87 cells and 15 days for SNB19.
8
Isolation and Cre-mediated Dicer Deletion in Pancreatic Acini
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Acinar units were isolated as described previously [24] (link). Briefly, mouse pancreata were perfused from common bile duct with 0.375 mg/ml Collagenase-P (Roche, 11213857001) in Hank’s balanced salt solution (HBSS). Subsequently, pancreata were harvested and incubated in 37°C water bath for 11 mins. Following multiple washes with HBSS and 5% FBS, hand-picked acini were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (Invitrogen), soybean trypsin inhibitor (Sigma) and antibiotics in low attachment 24 well plates (Corning). Acini were treated with either DMSO or with 4-hydroxytamoxifen (Sigma, H7904) at 20 ng/ml for 5 consecutive days to induce Cre-mediated deletion of Dicer in vitro.
9
Mammosphere Formation Assay Protocol
10
Quantifying Cell Death by ELISA
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Cells were plated on either cell culture-treated or low-attachment 24-well plates overnight (Corning, Manassas, VA, USA). Cell death was assessed by measuring histones and DNA fragmentation using the cell death ELISA, according to the manufacturer's instructions (Roche, Basel, Switzerland). Absorbance was normalized for cell number.
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