2.5 × 104 hepatoma cells were resuspended in 100 μL medium containing 1 % FCS and transferred onto 24-well cell culture inserts with a pore size of 8 μm (Corning, Tewksbury, USA). 600 μL medium containing 10 % FCS was added to lower chambers to generate a gradient for cell migration. Medium was removed and migrated cells on membranes were fixed with 4 % paraformaldehyde after 16 h. Cell nuclei of migrated cells at the bottom side of the membrane were stained with Hoechst 33342 (Life Technologies, Green Island, USA) and counted under the fluorescence microscope (Nikon, Tokyo, Japan). To analyze cell invasion, 24-well cell culture inserts were coated with rat-tail collagen (BD Biosciences, NJ, USA) prior to seeding of hepatoma cells. To examine transendothelial invasion, 2 × 105 HSECs were plated in 100 μL endothelial cell medium (Lonza, Basel, Switzerland) onto coated 24-well cell culture inserts and allowed to form a monolayer for 48 h. Subsequently, endothelial cell medium was aspirated and hepatoma cells were seeded in 100 μL medium containing 1 % FCS onto 24-well inserts. Transmigrated cells were visualized after 16 h and quantified as described for cell migration.
Endothelial cell medium (ecm)
Manufactured by Lonza
Sourced in Switzerland, United States
Endothelial cell medium is a specialized cell culture medium designed to support the growth and maintenance of endothelial cells. It provides the necessary nutrients, growth factors, and other components required for the optimal proliferation and differentiation of endothelial cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Hpmec, by Lonza (2 mentions)
Huvecs, by RIKEN BioResource Center (2 mentions)
Huvecs, by American Type Culture Collection (2 mentions)
Huvecs, by Lonza (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
24 well cell culture insert, by Corning (1 mentions)
Rat tail collagen, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by MultiSciences Biotech (1 mentions)
Phorbol 12 myristate 13 acetate, by MultiSciences Biotech (1 mentions)
Hpmec, by ScienCell (1 mentions)
Smbm medium, by Lonza (1 mentions)
Human epidermal growth factor (hegf), by Lonza (1 mentions)
Endothelial cell growth supplements, by ScienCell (1 mentions)
Hfgf b, by Lonza (1 mentions)
Penicillin streptomycin, by ScienCell (1 mentions)
Insulin, by Lonza (1 mentions)
Gentamicin amphotericin, by Lonza (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
11 protocols using endothelial cell medium (ecm)
1
Hepatoma Cell Migration and Invasion Assays
2
Cell Culture and Treatment Protocols
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The SK-N-SH (Non-MYCN amplification) and SK-N-BE(2) cell line (MYCN amplification) (obtained from the Shanghai Institute of Biological Cell Research, Chinese Academy of Sciences) was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS; Gibco) [22 (link), 23 ]. Additionally, the THP-1 cell line (also obtained from the Shanghai Institute of Biological Cell Research, Chinese Academy of Sciences) was cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/ml penicillin–streptomycin. HUEVC Human vascular endothelial cells were isolated by collagenase digestion of the human umbilical cord veins obtained from Children's Hospital Affiliated with Chongqing Medical University with the informed consents signed by donors. Human vascular endothelial cells were cultured in Endothelial Cell Medium (Lonza). 0.99 μm BKM 120 for SK-N-SH, 1.2 μm BKM 120 or SK-N-BE(2) (one of a PI3K inhibitor) (MCE)was used for the PI3K-AKT experiment [24 (link)]. For THP-1 cells, 100 ng/ml of PMA (Phorbol 12-myristate 13-acetate) (Multi-Science) was added, and recombinant human CXCL14 (MCE) was used for the experiment in vitro [25 (link)].
3
Culturing Human Pulmonary Endothelial and Smooth Muscle Cells
4
Isolation of Primary Renal Cells
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We recently produced GGTA1-KO and GGTA1/CMAH DKO pigs23 . Porcine kidneys from wild-type (WT), GGTA1-KO, and GGTA1/CMAH DKO pigs were flushed with 0.025% of collagenase type IV from Clostridium histolyticum (Sigma, St. Louis, MO) at 37 °C. Primary renal cell were isolated and cultured in Endothelial Cell Medium (Lonza, Basel, Switzerland). On day 3 post-isolation, cell sorting for CD31-positive (AbD SeroTec, Raleigh, NC) RMEC and AEC was performed using a flow cytometry cell sorter (BD Biosciences, cat.no.FACS Aria II, San Jose, CA). These cells were used within 3 to 5 passages.
5
Cell Culture Protocols for Vascular Research
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HUVECs were obtained from the Bioresource Collection and Research Center (BCRC) (Hsinchu, Taiwan) and grown at 37 °C in endothelial cell medium (Lonza, Walkersville, MD, USA) containing penicillin-streptomycin (1%) and endothelial cell growth supplement at 37 °C in an incubator containing 5% CO2. U937 cells were purchased from BCRC and grown in RPMI-1640 medium (Life Technologies; Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution at 37 °C in an incubator containing 5% CO2. A7r5 VSMC and RAW264.7 cells were purchased from BCRC and cultured in DMEM (Life Technologies; Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution at 37 °C in an incubator containing 5% CO2. Before conducting our experiments, the VSMCs were serum-starved for 24 h.
6
Culturing and Modulating Human Hepatoma Cells
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The human hepatoma cell lines 3p, 3sp, SNU-398, SNU-423, SNU-449 and SNU-475 were grown in RPMI-1640 medium plus 10 % fetal calf serum (FCS) and antibiotics. Hep3B, HepG2 and FLC-4 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM) plus 10 % FCS and antibiotics. PLC and HuH-6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10 % FCS and antibiotics. Human hepatic sinusoidal endothelial cells (HSECs) received endothelial cell medium (Lonza, Basel, Switzerland). All cells were kept at 37 °C and 5 % CO2. TGF-β signaling was stimulated by supplementing the medium with 2.5 ng/mL TGF-β1 (R&D Systems, Minneapolis, USA) for 24 h. For inhibition of TGF-β signaling, LY2109761 (Santa Cruz, Dallas, USA) antagonizing both TGF-β receptors I/II was used at a concentration of 10 μM for 24 h. All HCC cell lines were validated by short tandem repeat analysis.
7
Culture of Human Keratinocytes, Fibroblasts, and Endothelial Cells
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Human keratinocytes (human oral keratinocyte 1; OK1) were kindly provided by Dr. W-W Chang. The GM05386 cells (human primary fibroblasts) were obtained from the Coriell Cell Repository (Camden, New Jersey). The HUVECs were obtained from the Bioresource Collection and Research Center (BCRC). Human keratinocytes were cultured in DMEM containing 10% FBS, 1 mM pyruvate, 2 mM L-glutamate, and a 1% antibiotic/antimycotic solution. The GM05386 cells were cultured in DMEM containing 10% FBS and a 1% antibiotic/antimycotic solution at 37 °C in a 5% CO2 atmosphere. The HUVECs were grown in Endothelial Cell Medium (Lonza, Walkersville, MD, USA) containing penicillin-streptomycin (1%) and endothelial cell growth supplement at 37 °C in a humidified atmosphere of 95% air and 5% CO2 and were used between passages 2 and 5.
8
Isolation and Culture of Primary Murine Cells
9
Culturing Primary Endothelial Cells
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Primary human pulmonary microvascular endothelial cells (HPMECs) and human umbilical vein endothelial cells (HUVECs) obtained from Lonza (Walkersville, CA) were cultured in the Endothelial Cell Medium (ECM) with recommended supplements from the supplier and used in passages 3 to 5. The Madin-Darby canine kidney (MDCK) cell line, human sarcoma HeLa cells, HUVECs were all purchased from American Type Culture Collection (ATCC, Manassas, VA). They were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco, Gaithersburg, MA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher, Waltham, MA), penicillin-streptomycin (100U/ml, Thermo Fisher). Human lung epithelial cells A549 were purchased from ATCC, and cultured in RPMI 1640 (Thermo Fisher) with 5% FBS. Cells were incubated in a humidifier incubator at 37°C with 5% CO2.
10
Transwell Cell Migration Assay
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In the transwell cell migration assay, 24-well transwell plates with 8 μm pore size inserts (MCSP24H48, Millipore, Burlington, VT, USA) were used. HCAEC cells were re-suspended in an Endothelial Cell Growth Medium (ECM, Lonza Walkersville, Walkersville, MD, USA) without serum. A total of 2 × 105 cells were seeded onto the upper chamber (insert), while ECM supplemented with 5% FBS was added to the lower chamber, followed by incubation at 37 °C in 5% CO2 for 16 h. Then, a cotton tip was applied to remove the media and remaining cells that did not migrate down to the bottom side of the insert membrane. After that, the membranes were fixed with 100% methanol for 30 min, washed with PBS, and stained with crystal violet (Sigma, Burlington, VT, USA) for 30 min. For each insert, cells in the cross line forming 4 fields under a 100-fold magnification were counted. In addition to direct counting cells, we also repeated the assays with the stained cells that were collected and dissolved in 10% acetic acid and measured at a wavelength of 590 nm by the ELISA reader.
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