This study was approved by the ethics committee of the Siriraj Institutional Review Board (SIRB) (COA no. Si101/2015), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. The protocol for this study complied with all statements and principles set forth in the Declaration of Helsinki and all of its subsequent amendments, the Belmont Report, the CIOMS guidelines, and the ICH-GCP guidelines. Human chorionic tissue (hCr) was obtained from healthy newborns after receiving written informed consent to do so from their mothers. The hCr was cut into small pieces and incubated with 0.25% (w/v) trypsin–EDTA (GIBCO™; Invitrogen Corporation, Carlsbad, CA, USA) for 30 min at 37 °C. Cells were collected and washed with phosphate buffered saline (PBS) before being resuspended with culture medium and plated in culture vessels (Corning Incorporated, Corning, NY, USA). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The culture medium was replaced every other day.
Culture vessels
Manufactured by Corning
Sourced in United States
Culture vessels are used for culturing cells in a controlled environment. They provide a suitable substrate and conditions for the growth and maintenance of cell cultures. These vessels are designed to ensure optimal gas exchange, temperature, and humidity for cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Flp frt system, by Thermo Fisher Scientific (2 mentions)
Antibodies against strepii tag, by Novus Biologicals (2 mentions)
Low glucose dmem, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
T rextm 293 cells, by Thermo Fisher Scientific (1 mentions)
Schneider medium, by Thermo Fisher Scientific (1 mentions)
May grunwald giemsa, by Merck Group (1 mentions)
Flp in t rex 293 cell, by Thermo Fisher Scientific (1 mentions)
6 protocols using culture vessels
1
Isolation and Culture of Human Chorionic Cells
2
Umbilical Cord Mesenchymal Stem Cell Isolation
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Three umbilical cords (UC) were obtained, cut into small pieces, and incubated with 0.25% (w/v) Trypsin-EDTA (GIBCO™; Invitrogen Corporation, Carlsbad, CA, USA) for 30 min at 37 °C. Cell suspensions were collected and washed with phosphate-buffered saline (PBS) before being resuspended with culture medium, which consisted of Dulbecco’s modified Eagle’s medium (DMEM)-low glucose (Gibco®) supplemented with 10% fetal bovine serum (FBS; Merck Millipore, Burlington, MA, USA), and plated in culture vessels (Corning, Corning, NY, USA). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The culture medium was replaced every other day. Adherence cells were treated with 0.05% Trypsin-EDTA (Gibco®) and split to a seeding ratio of 5000 cells/cm2 for expansion.
3
Engineered Flp-In T-REx 293 Cell Lines
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Flp-InTM T-RExTM293 cells (Thermo Fisher, Cat#R78007) harboring IC2C wt or ∆ICN with C-terminal SNAP-FLAG-strepII tagwhich were generated using the FLP/FRT system (Thermo Fisher). Briefly, pcDNA5-FRT-TO construct and pOG44, which expresses Flipase, were co-transfected using Lipofectamine 2000 (Thermo Fisher) into Flp-InTM T-RExTM293 cells. After recovery from transfection, cells were grown in DMEM containing 10% FBS, 1% Penicillin-Streptomycin, and 100 µg/mL Hygromycin B to select cells in which pcDNA5 construct was inserted into FRT locus. The expression of tagged IC2C was verified by western blotting with antibodies against StrepII tag (Novus Biologicals). These cell lines were grown in culture vessels (Corning, Fisher Scientific) to 80% confluence, and then tagged IC2C expression was induced with 1 µg/ml doxycycline for two days. Cells were harvested in PBS by tapping the culture vessels, and spun down at 1200 rpm for 5 min in Sorvall Legend XTR. Cell pellets were washed with PBS, snap frozen in liquid nitrogen, and stored at −80 °C.
4
Quantifying Leishmania Infection in Macrophages
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Macrophages, cultured in RPMI 1640 culture medium containing 10% FBS, were incubated for 4 h with stationary-phase L. panamensis promastigotes at a 10:1 parasite-to-macrophage ratio. Then, cell monolayers were extensively washed and incubated in complete culture medium with or without edelfosine for 24 h. The intracellular parasite load was calculated by limiting dilution assay as previously reported [34 (link)]. Alternatively, macrophage monolayers infected with green fluorescent protein (GFP)-expressing p6.5-egfp-L. panamensis parasites were cultured in glass coverslips placed into culture vessels (Corning, Lowell, MA). After 24 h, coverslips were washed, and the rate of intracellular amastigotes and infected macrophages was visualized using a fluorescence microscope. Results are shown as the percentage of infected macrophages and as the parasite/macrophage ratio after counting 100 macrophages.
5
Quantifying Leishmania major Infection
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After 3 days, infected B-1CDP cells and macrophages monolayers were extensively washed, and medium was replaced by 0,5 ml of Schneider medium (Life Technologies), supplemented with 20% FCS and 2% human urine [34 (link)]. Monolayers were cultured at 26°C for additional 3 days. Intracellular load of L. major amastigote was estimated by production of proliferating extracellular motile promastigote in Schneider medium [34 (link)]. Alternatively, infected B-1CDP cells and peritoneal macrophages were cultured on glass coveslips place inside culture vessels (Corning) at 37°C in 7% CO2. After 4 days, coverslips were washed and stained with May-Grunwald Giemsa (Sigma-Aldrich), and intracellular amastigotes were counted in 100 B-1CDP cells or macrophages. Results are shown as amastigote number per phagocyte, and as percentage of infected macrophages. All results are mean and SE of triplicated cultures.
6
Flp-In T-REx 293 Cell Line Generation
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Flp-In™ T-REx™293 cells (Thermo Fisher) harboring IC2C wt or ΔICN with C-terminal SNAP-FLAG-strepII tagwhich were generated using the FLP/FRT system (Thermo Fisher). Briefly, pcDNA5-FRT-TOconstruct and pOG44, which expresses Flipase, were co-transfected using Lipofectamine 2000 (Thermo Fisher) into Flp-In™ T-REx™293 cells (Thermo Fisher). After recovery from transfection, cells were grown in DMEM containing 10% FBS, 1% Penicillin-Streptomycin, and 100 μg/mL Hygromycin B to select cells in which pcDNA5 construct was inserted into FRT locus. The expression of tagged IC2C was verified by western blotting with antibodies against StrepII tag (Novus Biologicals). These cell lines were grown in culture vessels (Corning, Fisher Scientific) to 80% confluence, and then tagged IC2C expression was induced with 1 μg/ml doxycycline for two days. Cells were harvested in PBS by tapping the culture vessels, and spun down at 1200 rpm for 5 min in Sorvall Legend XTR. Cell pellets were washed with PBS, snap frozen in liquid nitrogen, and stored at −80°C.
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