Authenticated human embryonic 293 kidney (HEK293) cells stably expressing the recombinant hFSHR (HEK293-hFSHR+) driven by the cytomegalovirus promoter [48 (link)], with less than 5 consecutive passages, were maintained in a humidified atmosphere of 5% CO2 at 37°C in high-glucose DMEM (Life Technologies Inc., Grand Island, NY) supplemented with 10% fetal calf serum (Sigma Aldrich, St. Louis, MO), 5 μg/mL geneticin (Life Technologies), and antibiotic (penicillin plus streptomycin) reagent (Life Technologies). Expression of the hFSHR protein at the cell surface plasma membrane was verified by immunoblotting as described later. Cells were grown to 70% to 80% confluence in 100-mm-diameter cell culture dishes (Corning, Corning, NY) at 37°C and an initial density of 1 × 106 cells. Twenty-four hours before the experiment, cells were mechanically dispersed and plated at a density of 75 or 200 × 103 cells/500 μL in 12- or 24-well culture dishes (Corning), depending on the particular experiment (see the following section). On the day of the experiment, cells were starved for 4 hours in DMEM-HEPES 10 mM medium (Life Technologies) supplemented with antibiotic and 0.1% BSA (Sigma). All experiments were performed in triplicate incubations, unless specified, and repeated at least 3 times.
24 well culture dishes
Manufactured by Corning
Sourced in United States
24-well culture dishes are a type of laboratory equipment used for cell culture and related experiments. They provide a standardized and controlled environment for growing and maintaining cells in a multi-well format. Each dish contains 24 individual wells, allowing for multiple experimental conditions or replicates to be tested simultaneously.
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Lab products found in correlation
Fetal calf serum (fcs), by Merck Group (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Mitotracker cmxros, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Fujifilm (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Axio observer, by Zeiss (1 mentions)
Matrigel, by Corning (1 mentions)
Matrigel substrate, by Corning (1 mentions)
Fibroblast growth factor (fgf), by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Cloning cylinder, by Merck Group (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
11 protocols using 24 well culture dishes
1
Stable Expression of Recombinant hFSHR in HEK293 Cells
2
Dexamethasone-Induced Thymocyte Apoptosis
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Thymocytes were plated in 24-well culture dishes (Corning) at 1.5 × 106 cells per well in cRPMI medium. Dexamethasone solubilized in DMSO (Sigma) was added to each well at final concentrations of 10−7 M and 0 M (vehicle only control) for each test sample. Cells were incubated 12 h at 37 °C, 5% CO2 in a humidified chamber. Post treatment, cells were collected into 5 mL round bottom tubes and centrifuged 400 × g, 5 min, RT. Cells were resuspended in FACS Buffer and stained with anti-CD4 and anti-CD8 antibodies. Post incubation 30 min, 4 °C, light safe, cells were washed in FACS Buffer and centrifuged as above. Apoptotic markers Annexin V and propidium iodide staining were completed using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, 556547) according to manufacturer’s protocol.
3
Mitochondrial Imaging and Immunofluorescence
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Non-sorted and sorted cells cultured with glucose or galactose were seeded in 24-well culture dishes (Corning) and incubated at 37 °C. The mitochondrial probes MitoTracker DeepRed and MitoTracker CMXRos were added at 2 and 10 nM, respectively, for 30 min at 37 °C (all from Invitrogen), followed by two washes with 1× PBS, and a final 5 min incubation with DAPI (2 mg/ml, Sigma). MitoTracker DeepRed was excited at 644 nm and the fluorescence emitted was detected at 665 nm (far red fluorescence) and assigned red fluorescence. MitoTracker CMXRos was excited at 579 nm and the fluorescence emitted was detected at 599 nm and assigned green fluorescence. For IF assays, cells were fixed with 4% PFA in PBS for 20 min at RT, washed with PBS, permeabilized with 1% Triton X-100 in PBS for 15 min, blocked with 1% BSA in PBS for 1 h at RT, and then incubated with specific antibodies (Supplementary Table 1 ) in a solution of 1% BSA in PBS. ProLong® Gold Antifade Reagent with DAPI (Cat no. P36941) was then added to mark cell nuclei. The fluorescent images were collected with a laser scanning confocal microscope Zeiss 710 and analyzed using the software Zen2009 5.5 (Oberkochen, Germany).
4
Generating MeWo Melanoma Cell Line Clones
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The human skin MeWo melanoma cell line was cultured in high-glucose Dulbecco’s
modified Eagle medium (DMEM; Fujifilm Wako Pure Chemical Corporation)
supplemented with 10% fetal bovine serum (FBS; Biological Industries) and a
penicillin-streptomycin solution (Thermo Fisher Scientific). MeWo cells were
seeded in 24-well culture dishes (Corning) 1 day before transfection. The
plasmid that encoded HS3ST4-cDNA with a pcDNA3 backbone or the vector plasmid
pcDNA3 (Thermo Fisher Scientific) was transfected in MeWo cells using FuGENE 6
Transfection Reagent (Promega). After incubation for 5 min at room temperature,
the mixture was added to the cells and incubated in a 5% CO2incubator at 37°C for 24 h, followed by medium exchange. Two days later, the
medium was changed to DMEM that contained 0.3 mg/mL G418 (Thermo Fisher
Scientific). Selection continued for 14 days. Before the end of selection, all
of the control MeWo cells without plasmids were dead. MeWo-HS3ST4 (+) and
MeWo-HS3ST4 (−) clones were then obtained by limiting dilution. We selected one
clone for use in the experiments. We repeated the experiments using another
clone and did not find significant differences between clones.
modified Eagle medium (DMEM; Fujifilm Wako Pure Chemical Corporation)
supplemented with 10% fetal bovine serum (FBS; Biological Industries) and a
penicillin-streptomycin solution (Thermo Fisher Scientific). MeWo cells were
seeded in 24-well culture dishes (Corning) 1 day before transfection. The
plasmid that encoded HS3ST4-cDNA with a pcDNA3 backbone or the vector plasmid
pcDNA3 (Thermo Fisher Scientific) was transfected in MeWo cells using FuGENE 6
Transfection Reagent (Promega). After incubation for 5 min at room temperature,
the mixture was added to the cells and incubated in a 5% CO2incubator at 37°C for 24 h, followed by medium exchange. Two days later, the
medium was changed to DMEM that contained 0.3 mg/mL G418 (Thermo Fisher
Scientific). Selection continued for 14 days. Before the end of selection, all
of the control MeWo cells without plasmids were dead. MeWo-HS3ST4 (+) and
MeWo-HS3ST4 (−) clones were then obtained by limiting dilution. We selected one
clone for use in the experiments. We repeated the experiments using another
clone and did not find significant differences between clones.
5
Biocompatibility and Wound Healing Potential of Hydrogel
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The biocompatibility of the hydrogel and its performance in a wound model was evaluated to analyze changes in its biological behavior due to the radiation process. Dermal fibroblasts were isolated from human skin after aesthetic surgeries and obtaining the signed informed consent. We used calcein and Ethidium homodimer 1 (EthD-1) to determine cell viability through the LIVE/DEAD Viability/Cytotoxicity for Mammalian Cells kit. Photographs were taken using an epifluorescence microscope AxioObserver (Zeiss ® , Germany). The number of live (calcein-positive) and dead (EthD-1-positive) cells were counted employing ImageJ software. For the wound-healing assay, fibroblasts were cultured until confluence in 24-well culture dishes (Corning ® , USA). Then, a scratch was performed using a 1-mL micropipette tip. Cells were cultured with the hydrogel (1, 2, 4, and 8%) in addition to the control condition (culture medium) and the vehicle condition (PBS, GIBCO ® , USA). The closure of the wound was analyzed for two days, and the distance between the cells was calculated using ImageJ software.
6
Extravillous Trophoblast Migration Assay
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Small 2-3 mm tissue sections were obtained from the tips of first-trimester human placental villi (6-10 weeks), dissected, and explanted in 24-well culture dishes pre-coated with phenol red-free Matrigel® substrate (Corning Life Sciences, New York, NY), as previously described 28 (link). Coated inserts were placed into 24-well culture dishes (Costar, Cambridge, MA). Villi explants were cultured in DMEM/F12 media containing 10% FBS. Placental villi that successfully anchored on Matrigel matrix and initiated outgrowth were used for the subsequent experiments, and are referred to as 24 h samples. EVT sprouting and migration from the distal end of the villous tips were recorded daily for up to 3 days. The extent of migration was measured via using ImageJ Pro 6.0 software. To test the effect of ALKBH5 on the migration of EVTs, 250 nM siRNA specifically targeting ALKBH5 or an equal concentration of control siRNA was introduced into two wells of extravillous explants from HCs or RM patients. The images were obtained after 24 h and 72 h of in vitro culture under a light microscope. Extravillous explants from HCs were incubated with lenti-ctrl or lenti-ALKBH5 lentiviral, and images after 24 h and 72 h of in vitro culture were taken under a light microscope. All explant experiments with cultured villi were repeated three times.
7
Placental Explant Culture to Assess Extravillous Trophoblast Migration
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Explant culture was performed as described previously 18 . In brief, small 2–3 mm tissue samples were obtained from the tips of first‐trimester human placental villi (8–10 weeks), dissected, and explanted in 24‐well culture dishes pre‐coated with phenol red‐free Matrigel substrate. Inserts were placed into 24‐well culture dishes (Costar, Cambridge, MA, USA). The explants were cultured in DMEM/F12 media with 5% FBS. Placental villi, anchored on Matrigel and successfully initiated to outgrow, were used for subsequent experiments and referred to as 24 h samples. EVT sprouting and migration from the distal end of the villous tips were recorded daily for up to 3 days. The extent of migration was measured at defined positions with the help of ImageJ Pro software. To test the effect of YY1 on the migration of EVTs, 100 nm siRNA specifically targeting YY1 or an equal concentration of control siRNA was introduced into two wells of culture media. Extravillous explants from patients with RM were incubated with lenti‐ctrl or lenti‐YY1 lentiviral contracts, and images after 24 and 72 h of in vitro culture were taken under a light microscope. All explant experiments with cultured villi were repeated three times. In each experiment, ten explants were analysed for both the YY1 siRNA and control groups (n = 10 in each group).
8
Cell Proliferation Assay with ACPSVs
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5 × 104 HeLa cells/well were seeded overnight in 24-well culture dishes (Costar) in regular DMEM media containing 10% FBS and Penicillin–Streptomycin antibiotics. The next day, the adherent cells were treated with media alone or media containing 50 µl of purified ACPSVs, which were purified from either healthy or apoptotic culture supernatants or from solid tumors. The proliferation-inducing effect associated with ACPSV contents was determined by cell counts after growing recipient cells for 24 h at 37 °C in the presence of 5% CO2, using a hemocytometer after trypsin digestion, as described4 (link).
9
First-trimester Placental Villous Explant Culture
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Small 2- or 3-mm tissue sections were obtained from the tips of first-trimester human placental villi (6–10 weeks), dissected, and explanted in 24-well culture dishes pre-coated with phenol red-free Matrigel substrate (Corning). Coated inserts were placed into 24-well culture dishes (Costar, Cambridge, MA). Villi explants were cultured in DMEM/F12 media containing 10% FBS. Placental villi that successfully anchored on Matrigel matrix and initiated outgrowth were used for the subsequent experiments and are referred to as 24 hr samples. EVT sprouting and migration from the distal end of the villous tips were recorded daily for up to 3 days. The extent of migration was measured via ImageJ Pro 6.0 software. Extravillous explants from RM patients were incubated with TTP-specific siRNA or control siRNA with 250 ng/mL, and images were obtained after 24 hr and 72 hr of in vitro culture under a light microscope. Ten explants per experiment were analyzed from both the TTP siRNA and control groups.
10
Sphere Formation Assay for Single PCa Cells
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Single PCa cells were resuspended in sphere culture medium (DMEM/F12 medium supplemented with 2% B27 (Gibco, 17504044), 1% N2 (Gibco, 17502001), 20 ng/mL fibroblast growth factor (PeproTech, 100-18B) and 20 ng/mL epidermal growth factor (PeproTech, AF-100-15-100), mixed at a 1:1 ratio with Matrigel (BD, 356234), and then seeded in 24-well culture dishes (Costar) at 1000 cells/well in a volume of 200 μL. Spheres formed were examined and photographed under a light microscope after 10 days. For serial sphere formation assay, spheres were harvested by centrifugation and digested with TrypLE for 5 min to obtain single cell suspensions. Secondary spheres were generated as described above.
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