Human hepatoma cells (HepG2) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone). Human T-cell leukemia cells (Jurkat) were maintained in RPMI-1640 supplemented with 10% FBS. Primary hepatocytes were isolated and cultured as previously described (Fan et al., 2020 (link)). Mouse Npnt promoter-luciferase constructs (Lanthier et al., 2011 (link)) and BRG1 expression constructs have been previously described (Chen et al., 2020c (link); Li et al., 2020a,b (link), c (link)). Small interfering RNAs were purchased from Dharmacon. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega) as previously described (Wu et al., 2020 (link); Yang et al., 2020a,b (link)). For conditioned media (CM) collection, the cells were switched to and incubated with serum-free media overnight. The next day, the media were collected, centrifuged at 4000 × g for 30 min at 4°C using 3-kDa MW cut-off filter units (Millipore) and sterilized through a 0.4-μm filter.
3 kda mw cut off filter units
Manufactured by Merck Group
Sourced in United States
The 3-kDa MW cut-off filter units are a type of laboratory equipment used for the separation and concentration of molecules based on their molecular weight. These filter units have a molecular weight cut-off of 3 kilodaltons, which means they can effectively retain molecules with a molecular weight greater than 3 kDa while allowing smaller molecules to pass through the filter.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase reporter assay system, by Promega (3 mentions)
Small interfering rna, by Horizon Discovery (3 mentions)
Angiotensin 2, by Merck Group (1 mentions)
Pfi 3, by Selleck Chemicals (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Ea hy926, by American Type Culture Collection (1 mentions)
Endothelial cell growth medium, by Lonza (1 mentions)
4 protocols using 3 kda mw cut off filter units
1
Hepatocyte and Leukemia Cell Culture Protocols
2
Endothelial Cell Characterization Protocol
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Immortalized human endothelial cells (EAhy926, ATCC), mouse macrophage-like cells (RAW264.7, ATCC), and HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone). Angiotensin II was purchased from Sigma. MRP8 promoter-luciferase constructs (Grebhardt et al., 2012 (link)) and BRG1 expression constructs (Li et al., 2019a (link)) have been previously described. Small interfering RNAs were purchased from Dharmacon. PFI-3 was purchased from Selleck. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega) as previously described (Li et al., 2019c (link)). For conditioned media (CM) collection, the cells were switched to and incubated with serum-free media overnight. The next day, the media were collected, centrifuged at 4000 × g for 30 min at 4°C using 3-kDa MW cut-off filter units (Millipore) and sterilized through a 0.4-μm filter.
3
Endothelial Progenitor Cell Conditioned Medium Protocol
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EPCs were cultured as described in a previous report with slight modification (Brunt et al., 2007). Bone marrow mononuclear cells were isolated from the bone marrow of 4-week-old female Sprague-Dawley rats by density gradient centrifugation, and these cells were incubated on culture dishes coated with fibronectin in endothelial cell growth medium (Lonza, MD, USA). To produce EPC-CM, EPCs were cultured for 48 hours under hypoxic conditions (1.5% O2, 5% CO2, 93.5% N2) in serum and growth factor-free endothelial cell basal medium (Lonza, MD, USA). The conditioned medium was collected and centrifuged at 4000 ×g for 30 minutes at 4°C using 3-kDa MW cut-off filter units (Millipore, Bedford, MA, USA), and then sterilized through a 0.4-µm filter. This concentrated medium (i.e., the EPC-CM) was stored at −80°C until use. Growth factor and serum-free endothelial cell basal medium served as control medium (Con-M).
4
Conditioned Media Collection Protocol
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Human hepatoma cells (HepG2) and mouse macrophage-like cells (RAW264.7) were maintained in DMEM supplemented with 10 % fetal bovine serum (FBS, Hyclone). Primary hepatocytes were isolated and cultured as previously described [44 (link)]. Human CCL7 promoter-luciferase constructs [45 (link)] and BRG1 expression constructs [46 (link)] have been previously described. Small interfering RNAs were purchased from Dharmacon. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega) as previously described [[47] (link), [48] (link), [49] (link), [50] (link)]. For conditioned media (CM) collection, the cells were switched to and incubated with serum-free media overnight. The next day, the media were collected, centrifuged at 4000×g for 30min at 4 °C using 3-kDa MW cut-off filter units (Millipore) and sterilized through a 0.4-μm filter.
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