Ectopic tissues were digested into the single-cell suspension with type IV collagenase (Solarbio, China). After centrifugation to pellet the cells, NanoShuttle (50 μl, Greiner bio-one Co., Germany) was added to the cell suspension, and incubated the cell-nano mix suspension was incubated at 37 °C for 1 h. After centrifugation to remove the supernatant, the number of cells was adjusted to 8*104/150ul with the medium mix. The cells were inoculated into a 96 well microplate (cell-repellent surface, Greiner bio-one Co., Germany). Then we hold the microplate on a magnetic driver (Greiner bio-one Co., Germany). The cell balls were placed in a 37 °C, 5% cell incubator and incubated for 15 min, and then the magnetic driver was removed.
Cell repellent surface
Manufactured by Greiner
Sourced in Austria, Germany
The cell-repellent surface is a specialized laboratory equipment designed to prevent the adhesion and growth of cells on its surface. It functions by creating a physical barrier that inhibits cell attachment, promoting a non-adherent environment for cells. The core purpose of this product is to provide a controlled surface for various cell-based experiments and studies, where the prevention of cell attachment is a critical requirement.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (2 mentions)
Collagenase type 4, by Solarbio (1 mentions)
Nanoshuttle, by Greiner (1 mentions)
Image pro software, by Media Cybernetics (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Egm 2mv medium, by Lonza (1 mentions)
Guava easycyte flow cytometer, by FlowJo (1 mentions)
Low melting agarose, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
10 protocols using cell repellent surface
1
Magnetic Cell Suspension Formation
2
3D Spheroid Viability Assay
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HCT116 cells were cultivated in three-dimensional (3D) multicellular spheroids. Briefly, 100 μL of a solution of cells (0.5 × 106 cells/mL) was inserted in 96-well plates with a cell-repellent surface (Greiner Bio-One, Kremsmünster, Austria) and cultured in complete medium plus 3% Matrigel (BD Biosciences, San Jose, CA, USA). Spheroids with stable structures had formed after three days. Then, the spheroids were exposed to a range of drug concentrations for 72 h, after which the cell viability was quantified by alamarBlue assay as described above.
3
3D HepG2 Spheroid Drug Screening
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HepG2 cells were cultivated in 3D multicellular spheroids. Briefly, 100 μL of cell solution (0.5 × 106 cells/mL) was added to a 96-well plate with a cell-repellent surface (Greiner Bio-One, Kremsmünster, Austria) and cultured in complete medium plus 3% Matrigel (BD Biosciences, San Jose, CA, USA). Spheroids with stable structures formed after three days. Then, the spheroids were exposed to a range of drug concentrations for 72 h. The negative control received the vehicle that was used for diluting the complex tested. In the end of the experiment, morphological changes were examined by light microscopy (Olympus BX41, Tokyo, Japan) using Image-Pro software (Media Cybernetics, Inc. Silver Spring, USA), and cell viability was quantified by alamar blue assay as described above.
4
Spheroid-Induced Macrophage Polarization
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Spheroids were generated via the cell aggregation (or pellet culture) method, as described previously [24 (link),25 (link)]. In brief, to generate homotypic or heterotypic spheroids, H522 tumour cells and AG02603 fibroblasts (AGFB) were seeded alone or together at a ratio of 4:1 (tumour: fibroblast; 1 × 104 total cells) in complete medium into 96-well u-bottom plates with cell-repellent surface (Greiner Bio-One; Austria). Spheroid cultures were immediately incubated for 48 h at 37°C in an incubator with 5% CO2 until spheroid formation.
For phenotyping experiments, positively selected CD14+ PBMCs (1 × 105) were antibody stained and analysed by flow cytometry prior to culture with spheroids to determine the baseline (T = 0) myeloid phenotype. CD14+ PBMCs (4 × 104) were added to the spheroid cultures on day 2 of spheroid formation and incubated for 48 h alongside M1-like, M2-like, and unpolarised media control Mφs. Spheroid-induced Mφ polarisation was measured by flow cytometry. Viable myeloid cells were gated based on positive CD14 staining and negative Fixable Viability Dye eFluor780 staining (eBioscience; San Diego, CA, United States).
For phenotyping experiments, positively selected CD14+ PBMCs (1 × 105) were antibody stained and analysed by flow cytometry prior to culture with spheroids to determine the baseline (T = 0) myeloid phenotype. CD14+ PBMCs (4 × 104) were added to the spheroid cultures on day 2 of spheroid formation and incubated for 48 h alongside M1-like, M2-like, and unpolarised media control Mφs. Spheroid-induced Mφ polarisation was measured by flow cytometry. Viable myeloid cells were gated based on positive CD14 staining and negative Fixable Viability Dye eFluor780 staining (eBioscience; San Diego, CA, United States).
5
Fabrication of Multicellular Spheroids
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Adherent cultures of each cell type were detached from the culture vessels with 0.25% trypsin-EDTA (Life Technologies, Grand Island, NY) solution. Trypsin was neutralized with appropriate GM, and cells were counted by a hemocytometer. Each cell type was then diluted to a concentration of 2500, 5000, and 10,000 cells in 200 μl of appropriate GM. The cell suspension (200 μl) was then pipetted into a single well of a U-bottom 96-well microplate with a cell-repellent surface (Greiner Bio-One, Monroe, NC). For fabrication of MSC/HUVEC spheroids made of 50,000 cells, MSCs and HUVECs were combined at a ratio of 92:8. The microplates were then incubated at 37°C in a 5% CO2 humidified atmosphere. Spheroid formation was monitored daily on an EVOS FL cell imaging system (Life Technologies). For fabrication of GFP+ HDF/MSC coculture spheroids, 5000 cells were used in total, and GFP+ HDFs and MSCs were cocultured in ratios of 2:1 and 1:1 for 1 day. During the fabrication and culture of HUVECs spheroids, EGM-2MV medium (Lonza) was used.
6
Neutrophil C42 mAb Staining Protocol
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Neutrophils (1.5 × 106 cells) were plated in multi-well suspension culture plates with cell-repellent surface (Greiner bio-one Kremsmünster, Upper Austria), and incubated for 2 h without or with 1 μg/mL C-36 peptide (used as a positive control). Cells were then collected into FACS tubes, washed with PBS (500 g, 5 min), and fixed with 3% PFA (paraformaldehyde) for 15 min at RT. Afterwards, cells were washed 3 times with PBS + 2% FCS and permeabilized with 0.2% Triton X-100 or 15 min at RT. Staining was performed with Dylight-488 conjugated C42 mAb. Samples were measured on Guava easyCyte flow cytometer (TX, USA) and data analyzed with FlowJo v10 (NJ, USA). Unlabeled cells were used as controls to set the flow-cytometry parameters.
7
3D Multicellular Spheroids Cytotoxicity Assay
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HCT116 cells were cultivated in 3D multicellular spheroids. Briefly, 100 μL of a solution of cells (0.5 × 106 cells/mL) were inserted in 96-well plate with a cell-repellent surface (Greiner Bio-One; Kremsmünster, Austria) and cultured in complete medium plus 3% matrigel (BD Biosciences; San Jose, CA, EUA). Spheroids with stable structures had formed after three days. Then, the spheroids were exposed to a range of drug concentrations for 72 h, after which the cell viability was quantified by alamar blue assay as described above.
8
Spheroid Formation Kinetics Assay
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Spheroid formation was assessed by seeding 5 × 103 cells/well in a 96-well plate with cell-repellent surface (Greiner Bio-One, Frickenhausen, Germany). Photomicrographs were taken after 24, 48, and 72 h of incubation at 37 °C and 5% CO2 in a humidified atmosphere.
9
Clonogenic and Spheroid Assays
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For soft agar assay, cells were seeded in 0.4% low-melting agarose (Sigma-Aldrich) in 6-well plate at a concentration of 5×10 3 cells per well. The cells were incubated at 37°C for 2-3 weeks, then stained with 0.005% crystal violet for 1 hour at room temperature before being destained in ddH 2 O.
In spheroid formation assay, cells were seeded in 96-well microplate (U-bottom) with cell-repellent surface (Greiner Bio-One GmbH, Frickenhausen, Germany) at a concentration of 2×10 3 cells per well. Cells were incubated at 37°C in a final volume of 200 µL culture medium per well for 2 weeks.
The volume of spheroids were monitored by inverted microscopy and calculated by use of the modified ellipsoid formula 1/2(length × width 2 ).
In spheroid formation assay, cells were seeded in 96-well microplate (U-bottom) with cell-repellent surface (Greiner Bio-One GmbH, Frickenhausen, Germany) at a concentration of 2×10 3 cells per well. Cells were incubated at 37°C in a final volume of 200 µL culture medium per well for 2 weeks.
The volume of spheroids were monitored by inverted microscopy and calculated by use of the modified ellipsoid formula 1/2(length × width 2 ).
10
Fusion of Heterogeneous Spheroids
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To assess spheroid fusion into larger constructs, two different methods were used: fusion in suspension and fusion in a hydrogel.
In the first method, HUVEC/HFF, HUVEC/ADSC and HUVEC/HFF/ADSC spheroids, cultured up to day 1 or day 10, were fused in suspension and fused constructs were compared.
Approximately 80 spheroids in 20 µl EGM-2 medium were seeded in U-shaped wells of a 96 well culture plate with cell-repellent surface (Greiner). After seeding, extra EGM-2 medium was added carefully to each well. Fused spheroids were harvested after 24 and 96 hours. For the second method, fusion of day 1 HUVEC/HFF/ADSC spheroids in a hydrogel was achieved. Hydrogels (Matrigel, Corning) of 100 µl, containing approximately 2000 spheroids, were generated. Matrigel, used at a final concentration of 10 mg/ml, was kept on ice while spheroids were harvested. After centrifugation, supernatant was removed and Matrigel was pipetted onto the spheroids. The solution was resuspended and 100 µl of the Matrigel containing spheroids was pipetted per well of a 96 well culture plate. After incubation at 37°C for 30 min, EGM-2 medium was carefully applied. Fusion of the spheroids in suspension and in Matrigel was evaluated by light microscopy up to 96 hours of culture.
In the first method, HUVEC/HFF, HUVEC/ADSC and HUVEC/HFF/ADSC spheroids, cultured up to day 1 or day 10, were fused in suspension and fused constructs were compared.
Approximately 80 spheroids in 20 µl EGM-2 medium were seeded in U-shaped wells of a 96 well culture plate with cell-repellent surface (Greiner). After seeding, extra EGM-2 medium was added carefully to each well. Fused spheroids were harvested after 24 and 96 hours. For the second method, fusion of day 1 HUVEC/HFF/ADSC spheroids in a hydrogel was achieved. Hydrogels (Matrigel, Corning) of 100 µl, containing approximately 2000 spheroids, were generated. Matrigel, used at a final concentration of 10 mg/ml, was kept on ice while spheroids were harvested. After centrifugation, supernatant was removed and Matrigel was pipetted onto the spheroids. The solution was resuspended and 100 µl of the Matrigel containing spheroids was pipetted per well of a 96 well culture plate. After incubation at 37°C for 30 min, EGM-2 medium was carefully applied. Fusion of the spheroids in suspension and in Matrigel was evaluated by light microscopy up to 96 hours of culture.
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