293T, HeLa, GT1–7, and MDCK cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). 293T cells were then transfected with Polyethylenimine (PEI), and HeLa, GT1–7, and MDCK cells were transfected with Genjet plus DNA in vitro transfection reagent (Signagen) following the manufacturer’s manual. The growth media of the transfected cells were replaced with FreeStyle serum-free media (GIBCO BRL) after 6h of transfection, and were collected as described in Figure 1A . 500 μl of the media were added into each well of the GE Whatman Dot-Blot 96 Well Plate System, which was applied with vacuum to aspirate the media across the PVDF membrane. The membranes, which captured proteins and nuclei acids in the growth media, were then blotted with anti-V5 and anti-GFP antibodies. In parallel, the transfected cells were lysed in RIPA buffer (PBS with 0.1% SDS), and supernatants were collected for western blot analyses.
Freestyle serum free media
Manufactured by Thermo Fisher Scientific
FreeStyle serum-free media is a cell culture medium designed for the growth and maintenance of various cell types in suspension culture. It is a chemically-defined, animal component-free formulation that provides the necessary nutrients and growth factors to support cell proliferation and viability without the use of serum.
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Lab products found in correlation
2 protocols using freestyle serum free media
1
Protein and Nucleic Acid Detection in Cell Culture Media
2
Protein and Nucleic Acid Detection in Cell Culture Media
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293T, HeLa, GT1–7, and MDCK cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). 293T cells were then transfected with Polyethylenimine (PEI), and HeLa, GT1–7, and MDCK cells were transfected with Genjet plus DNA in vitro transfection reagent (Signagen) following the manufacturer’s manual. The growth media of the transfected cells were replaced with FreeStyle serum-free media (GIBCO BRL) after 6h of transfection, and were collected as described in Figure 1A . 500 μl of the media were added into each well of the GE Whatman Dot-Blot 96 Well Plate System, which was applied with vacuum to aspirate the media across the PVDF membrane. The membranes, which captured proteins and nuclei acids in the growth media, were then blotted with anti-V5 and anti-GFP antibodies. In parallel, the transfected cells were lysed in RIPA buffer (PBS with 0.1% SDS), and supernatants were collected for western blot analyses.
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