This study was approved by the Research Ethics Board of Nanjing Drum Tower Hospital. Written consent was obtained from the parents. Before delivery, maternal blood was harvested for HBV serological screening examination. After full-term births, fresh UC was aseptically stored in sterile PBS and processed within 4 h after partum. HUMSCs were isolated from UC using the tissue explant method [37 (link)]. Briefly, the major blood vessels in fresh UC were removed in a laminar flow clean cabinet. After washing three times with 1 × PBS, the remaining connective tissue (Wharton’s jelly) was cut into small pieces of approximately 1 mm3. The tissue pieces were placed in a T75 cell culture flask (Corning, NY, USA) and kept in a 5% CO2 incubator at 37 °C without medium for 4 h. Then, 5 ml DMEM medium with 10% fetal bovine serum (FBS) was gently added to the culture flask. The culture medium was changed every 3–4 days. When well-developed colonies of fibroblast-like cells appeared, the cells were trypsinized and transferred into a new flask for further expansion. The cells were passaged three times to remove the contamination of other type cells and MSCs at the third passage were used for all of the experiments.
T75 cell culture flask
Manufactured by Corning
Sourced in United States
The T75 cell culture flask is a laboratory equipment designed for culturing cells. It provides a 75 cm² growth area for adherent cell lines. The flask is made of transparent polystyrene and features a vented cap to allow gas exchange. This product is intended for use in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Huvecs, by American Type Culture Collection (1 mentions)
24 well plate, by Corning (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Dmem medium, by Merck Group (1 mentions)
Humidified incubator, by Sanyo (1 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (1 mentions)
Il 17a, by R&D Systems (1 mentions)
Pneumacult ex plus basal medium, by STEMCELL (1 mentions)
Pneumacult ex plus 50x supplement, by STEMCELL (1 mentions)
Medium 106, by Thermo Fisher Scientific (1 mentions)
Pt 2501, by Lonza (1 mentions)
Pt 4106e, by Lonza (1 mentions)
35 protocols using t75 cell culture flask
1
Isolation of HUMSCs from Umbilical Cord
2
Culturing Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (CRL 1730). Cells were grown in minimum essential medium-L-glutamine (MEM), supplemented with 10% (v/v) fetal calf serum (FBS) and 1% penicillin-streptomycin-amphotericin (PSA). Cells were seeded in a T75 cell culture flask (Corning) and kept in a humidified incubator containing 5% CO2 at 37°C. The culture medium was replaced twice every week, and the cells were split 1 : 3 every week.
3
THP1 Macrophage Cytokine Response to Bacteria
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THP1 monocytes (1 × 106 cells/ml) were differentiated with 100 ng/ml Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) in T75 cell culture flask (Corning, New York, USA) for 48 h. Then PMA-differentiated THP1 macrophages were detached and resuspended in fresh culture medium at 1 × 106 cells/ml. 0.5 ml of cell suspension were seeded per well in 24-well plates (Corning, New York, USA) and cultured for another 24 h. Subsequently, culture medium were discarded and fresh medium containing various bacterial strains (bacteria to cells ratios of 40:1) or 1 μg/ml LPS (positive control; Invivogen) were added to cells. Un-stimulated cells served as negative control. After 24 h of stimulation, pro- and anti-inflammatory cytokines (IL-6 and IL-10) secretion in the supernatant were measured by ELISA (eBioscience, San Diego, USA) according to manufacturer’s protocol.
4
Culturing B16F10, L929, and BMDMs
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The cell lines, B16F10 and L929, were obtained from ATCC and cultured in RPMI 1640 medium (Corning incorporated, New York, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) as described previously (Liu and Yang, 2013 (link); Gao et al., 2017 (link); Phan et al., 2017 (link)). For the preparation of L929 supernatant, L929 were seeded into a T75 cell culture flask (Corning incorporated, New York, USA). After incubation for 8–10 days, cells were harvested and centrifuged to acquire the supernatant.
For bone-marrow derived macrophages (BMDMs) preparation, bone marrow cells (BMs) were flushed from femur and tibia bones of the mice. After the red cells were lysed, the BMs were grown in a humidified incubator at 37°C with conditional medium for 7 days containing 64% Dulbecco's modified eagle media (DMEM), 10% FBS, 1% penicillin-streptomycin, 25% L929 supernatant. Differentiated BMDMs were subjected to flow cytometry assays to determine the purity of macrophages. The percentages of macrophages (CD11b+F4/80+ cells) were more than 90%.
For bone-marrow derived macrophages (BMDMs) preparation, bone marrow cells (BMs) were flushed from femur and tibia bones of the mice. After the red cells were lysed, the BMs were grown in a humidified incubator at 37°C with conditional medium for 7 days containing 64% Dulbecco's modified eagle media (DMEM), 10% FBS, 1% penicillin-streptomycin, 25% L929 supernatant. Differentiated BMDMs were subjected to flow cytometry assays to determine the purity of macrophages. The percentages of macrophages (CD11b+F4/80+ cells) were more than 90%.
5
Culture and Expansion of C2C12 Myoblasts
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C2C12 myoblasts (CRL-1772) were acquired from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). Briefly, early passage cells were cultured in a DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) with a 10% fetal bovine serum (Gibco, ThermoFisher, Waltham, MA, USA) and 1% Anti-anti and Pen-Strep (ThermoFisher, Waltham, MA, USA) in a T75 cell culture flask (Corning Inc., Corning, NY, USA) under a humidified incubator (Sanyo, Osaka, JP) at 37 °C and 5% CO2. After the cells reached 75% confluence, subcultures were made using trypsin EDTA (Gibco, ThermoFisher, Waltham, MA, USA) 0.05% for 5 min. For the assessment, a 7 mL sample with 4 × 105 cells/mL were prepared.
6
Cell Fusion Assay for Retinal Damage
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Co-culture and cell fusion experiments were adapted from previous work in our laboratory.30 (link) To mimic damage conditions, human ARPE19 cells were grown in monolayer. In parallel, MGs pRLBP_eGFP and hMSC H2B::mRFP were cultured to 75–80% confluency. At day 1 of fusion, the ARPE19 monolayer was exposed to either 1 mM NMDA damage or PBS control. At day 0, cells were separately detached and co-cultured in suspension for 45 min, in a volume of 200 microliters, in a 1:1 ratio, with a maximum of 500,000 cells per cell fusion partner. Cells were then plated in a T75 cell culture flask (Corning) and left in culture overnight. On day 1, hybrids were detached and prepared for FACS cell sorting and for the following experiments.
7
Angiogenesis Assay with IL-17A
8
Expansion and Cryopreservation of hTBECs
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Primary normal human tracheal/bronchial epithelial cells, hTBECs, (Lonza, CC-2540S) were expanded in Pneumacult™-Ex Plus basal medium (STEMCELL Technologies, #05041) supplemented with Pneumacult™-Ex Plus 50X supplement (STEMCELL Technologies, #05042) and 0.5 mL of 200 mM hydrocortisone using the manufacturer’s recommended seeding density of 3,500 cells/cm2 on a T-75 cell culture flask (Corning). The cells were subcultured at the confluence of 70–80%. Upon collection, cells were passaged, used for organoid culture, or cryopreserved. Cells beyond passage number 4 were not used, as they did not yield adequate organoid formation.
9
Culturing Human Dermal Fibroblasts and Mesenchymal Stem Cells
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Human dermal fibroblasts, neonatal (HDFn, C0045C) were purchased from Thermo Fisher Scientific, USA. Cells were cultured in medium 106 (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin. The cells were maintained either in a T175 or T75 cell culture flask (Corning, USA) at 37 1C in a humidified incubator with 5% CO 2 . The cells were subcultured by using trypsin at approximately 80% confluency. The culture medium was replenished every 48 hours. Human bone marrow mesenchymal stem cells, (hMSCs, PT-2501) were purchased from Lonza, USA. Cells were cultured in medium (PT-4106E Lonza, USA) and supplemented with mesenchymal cell growth supplements (PT-4106E Lonza, USA), gentamicin sulfate Amphotericin-B (PT-4501E Lonza, USA) and L-glutamine (PT-4107E Lonza, USA). The cells were maintained either in a T75 or T150 cell culture flask (Corning, USA) at 37 1C in a humidified incubator with 95% air and 5% CO 2 . The cells were cultured by using trypsin at approximately 80% confluence. The culture medium was changed every 2-3 days.
10
Cultivation of Expi293F, LAD2, and Mast Cell Lines
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Expi293F cells (cat# A14527, Thermo Fisher Scientific, Waltham, MA, USA) were cultivated in 30 or 125 mL of Expi293 Expression Medium (cat# A1435101, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in 125 mL or 500 mL baffled Erlenmeyer flasks with vented caps (Corning, New York, NY, USA), respectively, at 37 °C, 8% CO2, according to the manufacturer’s instructions. LAD2 [31 (link)] (kind gift of A. Kirshenbaum and D. Metcalfe, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA) were grown in StemPro-34 medium (cat# 61870044, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) that was supplemented with 13 mL StemPro-34 Nutrient Supplement (cat# 10639011, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) per 500 mL medium, L-Glutamine (2 mM), penicillin (100 U/mL)/streptomycin (100 µg/mL), and rhSCF (100 ng/mL; cat#11343327, Immunotools GmbH, Friesoythe, Germany) in T75 cell culture flasks (Corning, New York, NY, USA) at 37 °C, 5% CO2. RBL-SX38 [34 (link)], RPMI-8866 (ECACC 95041316), and U937 cells (ATCC® CRL-1593.2™) were cultivated in RPMI 1640 medium with GlutaMAXTM (cat# 618700, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and maintained in T75 flasks (Corning, New York, NY, USA) at 37 °C, 5% CO2.
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