Chondrogenic induction was performed according to previous published protocol (Nantavisai et al., 2020 (link)). Cells were seeded at the density of 5 × 104 cells per well in a 24-well culture plate (Corning, USA) and further maintained in chondrogenic induction medium for 21 days. Chondrogenic induction medium contained DMEM supplemented with 1% Glutamax (Thermo Fisher Scientific, USA), 1% antibiotic-antimycotic (Thermo Fisher Scientific, USA), 50 mg/mL L-ascorbic-2-2phosphate (Sigma-Aldrich, USA), 40 mg/mL L-proline (Sigma-Aldrich, USA), 0.1 μM dexamethasone (Sigma-Aldrich, USA), 1% insulin-transferrin-selenium (Thermo Fisher Scientific, USA), 10 ng/mL transforming growth factor beta 3 (TGF-β3) (Sigma-Aldrich, USA), and 15% FBS (Thermo Fisher Scientific, USA). The medium was changed every 48 h. At day 21, a 0.1% alcian blue solution (Sigma-Aldrich, USA) was used to stain acidic polysaccharide. Chondrogenic marker gene expression was analyzed by RT-qPCR.
24 well culture plate
Manufactured by Corning
Sourced in United States, Switzerland
The 24-well culture plates are a versatile laboratory equipment designed for cell culture and assay applications. These plates provide 24 individual wells for growing and maintaining cells or conducting various experiments. The wells have a standardized size and shape to ensure consistent results and ease of use.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Matrigel, by BD (9 mentions)
Dexamethasone (dex), by Merck Group (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Non essential amino acid (neaa), by Cambrex (4 mentions)
Sodium pyruvate, by Merck Group (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Annexin 5 apoptosis detection kit 1, by BD (3 mentions)
β glycerophosphate, by Merck Group (3 mentions)
Insulin, by Merck Group (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Powershot a640, by Canon (3 mentions)
Axiovert 40 cfl inverted microscope, by Zeiss (3 mentions)
M csf, by Thermo Fisher Scientific (3 mentions)
200 protocols using 24 well culture plate
1
Chondrogenic Induction of Stem Cells
2
Retinoic Acid Effects on Feline Follicle Development
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To determine the effect of RA on in vitro follicle development, ovarian cortical pieces from prepubertal (n = 9) versus adult (n = 13) cats were incubated separately for 7 d on 1.5% (w/v) agarose gel blocks in protein-free medium within a 24 well culture plate (Corning Incorporated, Corning, NY), as described previously [12 (link)]. The ovarian cortical pieces combined from both ovaries were randomly divided into fresh, non-cultured control or three culture treatment groups. The culture medium (as described above) was supplemented with 0 (control), 1 or 5 μM RA (2–5 cortical pieces/cat/treatment). Cortical pieces of each donor cat were assessed for viability on the same day of sample collection (Day 0) or after 7 d of culture.
3
GMSC Modulation of T Cell Apoptosis
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WT GMSCs, FasL-/- GMSCs, or FasL overexpressed FasL TF GMSCs (2 × 105) were seeded on a 24-well culture plate (Corning) containing Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland) with 10 % heat-inactivated FBS, 50 μM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate (Sigma-Aldrich), 1 % non-essential amino acid (Cambrex, East Rutherford, NY, USA), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. After incubation for 24 h, T lymphocytes (1 × 106) from the spleen, pre-stimulated with plate-bound anti-CD3e (3 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies, were directly loaded onto GMSCs and co-cultured for 2 days. Apoptotic T cells were detected by staining with CD3 antibody, followed by the AnnexinV Apoptosis Detection Kit I (BD Bioscience), and then analyzed by FACS Calibur flow cytometer.
4
Co-culture of CAFs and Pancreatic Cancer Cells
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The co-culture assay was performed in a non-contact manner, using a Transwell with 0.4-µm pores (Corning, Inc.). First, CAFs (1.0×104 per well) were plated on the top layer and incubated overnight in DMEM containing 10% FBS. Once the CAFs had settled in the Transwell, PSN-1 cells (a human PC cell line, p53 mutant) were plated at 1.0×105 per well on the lower layer of a 24-well culture plate (Corning, Inc.). PSN-1 cells were obtained from the American Type Culture Collection. After an additional 48 h of co-culture in DMEM containing 0.5% FBS, the Transwell was removed and PSN-1 cell proliferation was evaluated.
5
Fibroblast Cell Growth Assay
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The cell growth assay was performed using a method reported elsewhere.27 (link) Briefly, fibroblasts (25,000 cells) in 2 mL of DMEM 10% were allowed to settle in a well of a 24-well culture plate (Corning Corporation, Cambridge, MA, USA) for 2 hours. After slight washing with PBS, the medium was replaced by DMEM containing 1.0% fetal bovine serum (DMEM 1%) for 12 hours. Cells treated with TGF-β1, hydrogel carrying TGF-β1, and PBS (control) were harvested using 0.2% trypsin in PBS, and stained with Calcein-AM for live cell counting at an emission wavelength of 540 nm in a fluorescence microscope. Fluorescence intensities relating to the number of cells were recorded for analysis. All experiments were performed in triplicate.
6
Transwell Cell Migration and Invasion Assay
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Cells were suspended in serum-free DMEM. One hundred microliters of the cell suspension was seeded in the upper compartment of 8 µm transwell chambers in a 24-well culture plate (Corning, USA) for 48 h, and 500 µl of DMEM supplemented with 10% FBS was added to the lower compartment of the chambers. Chambers with inserts uniformly coated with Matrigel (BD Biosciences) were used for the invasion assay. The transwell chambers were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30 min at room temperature. Then, cells on the upper surface of the inserts were wiped off with a cotton swab. Stained cells were washed with PBS and observed with a microscope. The capacities for cell migration and invasion were assessed by determining the average numbers of stained cells in 5 regions.
7
Sphere-Forming Cells Protocol
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Sphere-forming cells were obtained as previously described (Ciccarelli et al., 2016 (link); Camero et al., 2020 (link); Megiorni et al., 2021 (link)). Briefly, RD and RH30 cells were cultured in anchorage-independent conditions (ultra-low attachment flasks or plates, Corning) in stem cell (SC)-medium consisting of DMEM:F12 medium (Gibco-Invitrogen) and B27 (ThermoFischer). Fresh human epidermal growth factor (20 ng/ml) and fibroblast growth factor (20 ng/ml) (PeproTech, London, United Kingdom) were added twice/week until cells formed floating spheres. To evaluate the primary sphere formation, cells from sub-confluent (70–80%) monolayer cultures were plated at a density of 100, 500 or 1,000 cells in a 24-well culture plate (Corning Inc., Corning, NY, United States). For the sphere formation assay, the number of primary tumor spheres was determined.
8
ELISPOT Assay with PBMC Stimulation
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Before the ELISPOT assay was performed, PBMCs were first stimulated with polyclonal activator R848 and recombinant human IL-2 (Mabtech AB, Stockholm University, Sweden). During the cell stimulation process, 1 × 106 cells per mL were seeded into the 24-well culture plate (Corning Inc.). In parallel to the stimulated samples, non-stimulated samples were also employed. All the samples were incubated at 37 °C in a humidified 5% CO2 incubator for 72 h.
9
Stem Cell-T Cell Apoptosis Assay
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Stem cells (0.2 × 106/per well) were seeded into a 24-well culture plate (Corning) which contained Dulbecco’s modified Eagle’s medium (DMEM; Switzerland) with 10% fetal bovine serum (FBS), 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate (Sigma-Aldrich), 10 mM HEPES, 1% nonessential amino acid (Cambrex, USA), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM l -glutamine. After 24-h incubation, T lymphocytes (1 × 106) from the spleen were treated with plate-coated anti-CD3ε (3 μg/mL) and anti-CD28 (2 μg/mL) antibodies, which were then co-cultured with stem cells for 2 days. CD3 antibody staining was used to detect apoptotic T cells, which was followed by the Annexin V Apoptosis Detection Kit (BD Biosciences, USA) detection.
10
Neutrophil Activation Kinetics with Anakinra
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For all assays, neutrophils were resuspended in Gibco RPMI 1640 media (Thermo Fisher, Waltham, MA, USA) containing 1% BSA (Sigma-Aldrich, Saint Louis, MO, USA). For the MPO and calcium measurements, the cells were seeded into white flat-bottom 96-well assay plates (Corning Incorporated, New York, NY, USA) at a density of 2 × 105 cells per well. Supernatant sample generation for NE and cfDNA assays was done by seeding cells in a 24-well culture plate (Corning Incorporated, New York, NY, USA), at a concentration of 1 × 106 cells per well. Cells were treated with concentrations of 100 ng/ml, 100 µg/ml and 1 mg/ml anakinra (Swedish Orphan Biovitrum AB, Stockholm, Sweden) at 60, 30, and 10 minutes prior, at the time of and 30 and 60 minutes after stimulation with 100 nM PMA or 5 µg/ml LPS (E.coli O55:B5) as indicated by other studies [25 (link), 27 (link)-29 (link)]. Stimulation was done for 3 hours.
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