Transwell invasion assay was performed using our modified protocol [57 (link),67 (link)]. Briefly: Nunc Cell Culture Inserts (transwell) with 8.0μm pore diameter (#141006) were covered with 50 µL 0.2% gelatin 1 h 37 °C. Next, gelatin was carefully removed. A549 or A549CisR cells were treated with or without 3 µM #19 for 24 h. Then, they were trypsinized, washed 2× with medium, and transferred (2 × 105 cells/chamber) to upper chamber of Nunc Cell Culture Inserts in 0.1% BSA medium—supplemented with or without 3 µM #19. Full medium in lower chamber was used as chemoattractant. Next, medium and the gelatin from the top surface of the membrane were removed, invaded cells on the bottom surface of the membrane were washed 2× with PBS and then fixed for 5 min with CellFIXTM. Cells were dyed at RT 15 min with Hoechst 33,342 (Molecular Probes/Life Technologies, Waltham, MA, USA). Finally, membranes were cut out from chambers and placed on microscope glass, and number of cells that migrate into the membrane was counted in 5 random spots.
Nunc cell culture inserts
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc™ cell culture inserts are designed for cell culture and transwells applications. They feature a transparent polyethylene terephthalate (PET) membrane with a defined pore size, enabling cell migration and invasion studies.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (2 mentions)
Cnt pr 3d medium, by CELLnTEC (2 mentions)
Epilife medium, by Thermo Fisher Scientific (2 mentions)
Rat tail collagen, by BD (2 mentions)
Penicillin streptomycin, by CELLnTEC (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Crystal violet solution, by Merck Group (1 mentions)
Dmire2, by Leica (1 mentions)
Human keratinocyte growth supplement, by Thermo Fisher Scientific (1 mentions)
8 protocols using nunc cell culture inserts
1
Transwell Invasion Assay of A549 Cells
2
Suppressing ALDH+ Cells in DOX-Treated SUM159
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Co-culture of DOX-treated SUM159-shXIST cells with ALDH− cells presorted from SUM159-shXIST or SCR cell lines in lower and upper chambers was conducted with Thermo Scientific™ Nunc™ cell culture inserts in carrier plate, 6 well format (Product code: 3491). Briefly, SUM159-shXIST cells pretreated with DOX (1 µg/mL) for 3 days were plated at 1 × 105/well in 6-well carrier plate 2 h before placing the Nunc™ cell culture inserts loaded with 4 × 105/well of ALDH− cells sorted from SUM159-shXIST or SCR cells. After incubation with DOX (1 µg/mL) containing media supplemented with or without IL-6 neutralizing antibody (final concentration at 200 ng/mL, Proteintech, Catalog No. 69001-1-Ig) for 4 days, SUM159-shXIST cells in each well of the carrier plate were harvested and analyzed for ALDH+ cell content using ALDEFLUOR Assay.
3
Patulin-Induced Neurite Outgrowth Quantification
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To quantitatively assess the neurite outgrowth after patulin-treatment of enteric neurons, a cellular transwell culturing system modified from [57 (link)] was used. Cell culture transwell inserts with a 3 micron pore size in 24-well formats (Nunc™ cell culture inserts in multi-well plate) were coated with extracellular matrix gel from Engelbreth-Holm-Swarm murine sarcoma (Sigma Aldrich, diluted 1:5 with culture medium). Drug concentration was set to 0.5 µM in 500 µL culture medium (100 µL cell suspension in insert and 400 µL culture medium in basal chamber). After an incubation period of 24 h, neuronal cell bodies from the upper surface of the insert were removed with a cotton swab. Neurites were subsequently stained with crystal violet solution (Sigma Aldrich). For extraction of the dye, inserts were transferred into 400 μL extraction buffer and shaken for 5 min at 600 rpm at room temperature. To quantify neurite outgrowth, 100 µL of extracted stain solution was measured at OD595 and normalized to OD340 (Biochrom ASYS Hitech Expert 96 UV Microplate Reader).
4
Transwell Assay for VEGF-Induced Cell Migration
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HT-29 colon cancer cells were seeded onto the polycarbonate filter of upper chamber of Nunc™ cell culture inserts in carrier plate systems in free medium with 10 ng/mL VEGF with or without Avastin or a series of concentrations of piceatannol, in parallel, the culture medium supplied with 10% FBS containing 10 ng/mL VEGF in the presence or absence of piceatannol was added into lower chamber. After incubated with drugs for 24 h, cells left on the top side of polycarbonate Transwell® filters were carefully removed by using cotton swabs. Cells migrated into opposite side were subjected to be on fixation for 15 min at room temperature by using 4% formaldehyde in 1× PBS. The nuclei of cells were stained by DAPI, and ten images were randomly captured for each well by using a fluorescence microscopy (DMIRE2, Leica, Wetzlar, Germany) equipped with Leica confocal software (Version 2.61), 10× objective. The wavelengths for excitation and emission were separately set at 358 nm and 461 nm. Total cell number of 10 images was counted for each well.
5
Generation of Human Epidermal Equivalents
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Human N/TERT keratinocyte cell line N/TERT-2G, purchased from J. Rheinwald laboratory (Harvard Medical School, Boston, MA, USA), was cultured in Epilife medium (MEPI500CA, ThermoFisher Scientific, Waltham, MA, USA), complemented with human keratinocyte growth supplement (S0015, ThermoFisher Scientific) and 1% penicillin/streptomycin (P4333, Sigma-Aldrich, Saint-Louis, MO, USA). Human epidermal equivalents (HEEs) were generated as previously described62 (link), with minor adjustments. Briefly, inert Nunc cell culture inserts (141002, ThermoFisher Scientific) were coated with rat tail collagen (100 μg/mL, BD Biosciences, Bedford, USA) at 4°C for 1 hour. 1.5×105 N/TERT-2G keratinocytes (either wildtype, ΔAHR, or ΔTFAP2A keratinocytes) were seeded on the transwells in 150 μL Epilife medium (ThermoFisher Scientific) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich) in a 24 wells format. After 48 h, cultures were switched to a mixture of CnT-PR-3D medium (CELLnTEC, Bern, Switzerland) and DMEM medium (60:40 (v/v)) without penicillin/streptomycin for 24 h and then cultured at the air-liquid interface for an additional ten days. Culture medium was refreshed every other day until harvesting at day ten of the air-exposed phase.
6
Cell Migration and Invasion Assay
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Cell migration was analyzed using microwell inserts (pore size, 8-µm; Nunc™ Cell Culture Inserts; Thermo Fisher Scientific, Inc., Waltham, MA, USA), while cell invasion was analyzed using Corning® BioCoat™ Growth Factor Reduced Matrigel Invasion Chambers (pore size, 8-µm; BD Biosciences, Franklin Lakes, NsJ, USA). For the two assays, culture medium supplemented with 20% heat-inactivated foetal calf serum was used as a chemoattractant in the lower reservoir. All measurements were performed in three independent experiments using sequential treatment with aza and TSA. Negative controls were performed using acetic acid and dimethyl sulfoxide alone. Subsequent to 72 h, pre-treated cells were harvested, washed and seeded in the upper reservoir. After 24 h, cells on the lower side of the membrane were counted following staining with Giemsa. All values were corrected against the proliferation ratio of BrdU (aza + TSA) / BrdU (control).
7
Caco-2 Cell Culture and Transport Studies
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Caco-2 cells were grown to 50–60% confluence in DMEM Glutamax cell culture medium, which contained 4 µg/ml folate (specifications) and no vitamin B12 (specifications and measurements), as far as the cofactors of one carbon metabolism were concerned. The medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS; 20 pM B12 in the original serum), 100 U/ml penicillin, and 100 µg/ml streptomycin. The other conditions included humidified 5% CO2/95% air atmosphere at 37°C. The culture medium was replaced every second day. The cells were subcultured by trypsinization with 0.25% trypsin and 0.9 nM EDTA in Dulbecco’s phosphate-buffered saline (PBS) without Mg2+ or Ca2+ (DPBS–).
The Caco-2 cells used for transport studies were seeded in Nunc cell culture inserts (growth area 3.14 cm2, pore size 0.4 µm; Thermo Fisher Scientific, Waltham, MA) at a density of ∼2.25 × 105 cells/insert. The seeded cells were cultured in growth medium (1 ml apically and 2 ml basolaterally) with changes of medium 16 h after seeding and then every second day.
The Caco-2 cells used for transport studies were seeded in Nunc cell culture inserts (growth area 3.14 cm2, pore size 0.4 µm; Thermo Fisher Scientific, Waltham, MA) at a density of ∼2.25 × 105 cells/insert. The seeded cells were cultured in growth medium (1 ml apically and 2 ml basolaterally) with changes of medium 16 h after seeding and then every second day.
8
Generation of Epidermal Equivalents for CRISPR Editing
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Epidermal equivalents were generated as previously described (Smits et al., 2017) , with minor adjustments. Briefly, inert Nunc cell culture inserts (141002, Thermo Fisher Scientific) were coated with rat tail collagen (100 mg/ml, BD Biosciences, Bedford, MA) at 4 C for 1 hour. A total of 1.5 Â 10 5 N/TERT-2G KCs were seeded on the transwells in 150 ml Epilife medium (Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich) in a 24wells format. After 48 hours, cultures were switched to a mixture of CnT-PR-3D medium (CELLnTEC, Bern, Switzerland) and DMEM medium (60:40 [v/v]) without penicillin/streptomycin for 24 hours and then cultured at the airÀliquid interface for an additional 10 days. The culture medium was refreshed every other day until harvesting on day 10 of the air-exposed phase.
sgRNA design, single-strand donor oligonucleotide, and synthetic Cas9
Synthetic sgRNAs to knockout FLG gene and purified Edit-R Cas9 nuclease protein (nuclear localization signal, number CAS11200) were obtained from Synthego (Menlo Park, CA) and IDT Technologies (Coralville, IA), respectively. Custom synthetic Alt-R sgRNAs and single-strand donor oligonucleotide to correct FLG expression were ordered from IDT Technologies. See Supplementary Table S4 for details on the sgRNAs and single-strand donor oligonucleotide used.
sgRNA design, single-strand donor oligonucleotide, and synthetic Cas9
Synthetic sgRNAs to knockout FLG gene and purified Edit-R Cas9 nuclease protein (nuclear localization signal, number CAS11200) were obtained from Synthego (Menlo Park, CA) and IDT Technologies (Coralville, IA), respectively. Custom synthetic Alt-R sgRNAs and single-strand donor oligonucleotide to correct FLG expression were ordered from IDT Technologies. See Supplementary Table S4 for details on the sgRNAs and single-strand donor oligonucleotide used.
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