Cell culture methods and transient transfection protocols were used as previously published (3). Briefly, HEK-293F (HEK293F) FreeStyle (Invitrogen) were grown in serum-free FreeStyle 293 expression medium (Invitrogen) with shaking at 130 rpm in a 37 °C incubator under 8% CO2. The HEK293F cells were transfected using 2 ml of OptiMem-I reduced serum medium containing 40 µl of HEK293Fectin reagent (Invitrogen) with 20 µg of each expression plasmid. Cells were harvested after 48 h transfection. In some experiments, all-trans retinol (Sigma, St. Louis, MO) was added 24 h post-transfection to a final concentration of 2.5 µM, the cells were cultured for a further 7 h, and then harvested by centrifugation for retinoid analysis.
Opti mem 1 reduced
Manufactured by Thermo Fisher Scientific
Opti-MEM I reduced is a cell culture medium designed to maintain cell viability and growth in a serum-reduced environment. It is a modification of the original Opti-MEM I medium, with a reduced concentration of certain components. The core function of Opti-MEM I reduced is to provide a balanced salt solution and essential nutrients to support the survival and proliferation of cells in culture.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (1 mentions)
Gfp lc3 plasmid, by Cell Biolabs (1 mentions)
Sc 40986, by Santa Cruz Biotechnology (1 mentions)
Sc 37007, by Santa Cruz Biotechnology (1 mentions)
Amaxa cell line nucleofector kit l, by Lonza (1 mentions)
6 protocols using opti mem 1 reduced
1
HEK293F Cell Culture and Transfection
2
Overexpressing TFEB Modulates Cd Toxicity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to the manufacturer’s instructions, cells were transfected with Opti-MEM® I reduced serum media and Lipofectamine 2000 (Invitrogen, 11668–019). For Tfeb overexpression, two plasmids, pEGFP-N1-TfebS141A, pEGFP-N1-TfebS141D, and control plasmids designed by Invitrogen Corporation (Shanghai, China) as well as GFP-LC3 plasmid (Cell Biolabs, CBA-401) were transfected into Neuro-2a cells. At 24 h after transfection, the cells were exposed to 50 μM Cd for 24 h.
3
HepG2 SIRT1 knockdown protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 cells grown in antibiotic-free DMEM on culture dishes for 24 h were transfected with SIRT1 small-interfering (siRNA; 80pmols; sc-40986, Santa Cruz, CA) and non-targeted control siRNA (sc-37007, Santa Cruz, CA) using Opti-MEM I reduced serum media and Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, CA). Twenty-four hours after transfection, cells were washed and processed for immunoblotting and other studies as described below.
4
Redox Imaging with PF-H2TMRos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Redox-sensor Red (PF-H2TMRos; Life Technologies) at a final concentration of 5 μM in Opti-MEM I reduced serum culture medium (phenol red-free; Invitrogen) was loaded into cells for 10 min and cells were imaged in fresh medium as described previously [24 (link)].
5
Modulating Stem Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A pool of 3–4 individual siRNA probes were used to knockdown targets at a final concentration of 100 nM. siRNA probes were dissolved in 1.5% Lipofectamine RNAiMAX (Invitrogen, 13778150) in Opti-MEM I reduced serum medium (Invitrogen, 31985062). For 96-well plates, 100 μl primary cells (25 × 104 ml−1) were reverse-transfected with 20 μl 100 nM of corresponding siRNA. At 48 h after transfection, cells were changed to culture medium without siRNA. For the Transwell co-culture experiment, eP3 cells were cultured in inserts with corresponding siRNA for 48 h with a blank receiver plate. At 48 h after transfection, the inserts were washed with warm PBS twice and co-cultured with Lin−Sca1+CD142− cells growing on receiver plates until the end of the experiment. For the β-catenin experiment, 48 h after transfection, eP1 cells were changed to culture medium supplemented with 0.5 μg ml−1 recombinant RSPO2 for 24 h.
6
Transient and Stable Gene Silencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transient gene silencing was performed by using human β-arrestin 2 validated siRNA and a non-specific-control siRNA (Custom SMARTpool siRNA Design Service of Dharmacon, Inc., Lafayette, CO). Transfection was performed using Lipofectamine RNAiMAX and Opti-MEM I reduced serum medium according to the manufacturer's instructions (Invitrogen). Western blot analysis was performed to confirm silencing.
RNA interference was also performed by an Amaxa cell line Nucleofector kit L (Lonza, Basel, Switzerland) [26] using two shRNA plasmid constructs (10 µg/10 6 cells) for human CXCR7 to obtain long term silencing. A control non-targeting (NT) shRNA plasmid construct expressing an shRNA sequence with no significant homology to any
RNA interference was also performed by an Amaxa cell line Nucleofector kit L (Lonza, Basel, Switzerland) [26] using two shRNA plasmid constructs (10 µg/10 6 cells) for human CXCR7 to obtain long term silencing. A control non-targeting (NT) shRNA plasmid construct expressing an shRNA sequence with no significant homology to any
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!