Navigation Links
General Considerations for Successful Transfection Experiments

Cells

General
Keep an eye on your cells; ensure they are in good condition and set a suitable plating protocol for optimal cell density from the beginning to the end of transfection.

Dividing versus Non-dividing Cells
Dividing cells tend to be more accessible for uptake and expression of foreign DNA compared to quiescent cells. Mitogenic stimuli (e.g., virus transformation, growth factors, conditioned media, and feeder cells) are often used to activate primary cells.

Adherent versus Suspension Cells
Transfection efficiencies differ significantly between adherent and suspension cells. It is speculated that the limiting step is the uptake by endocytosis; however, a plausible mechanistic explanation on a molecular level does not yet exist. Therefore, the search for more efficient transfection reagents is mainly empirical.

Splitting Protocol
Before splitting, adherent cells must be trypsinated in order to remove them from the substrate. This routine step means a severe obstruction of normal cellular functions. Differences in the splitting protocol (e.g., extension of trypsination, inactivation of trypsin, and time until transfection starts) could have an impact on transfection experiments.

Passage Number
Cell lines tend to be unstable. As a result, their features may change over time in culture. The passage number indicates how often a cell line has been split (normally within one lab). In most cases, the exact passage number existing prior to establishment of the line is unknown. Different culture conditions could lead to clonal selection. Cell lines with the same name could thus significantly differ with respect to physiology and morphology (and transfectability). Generally, cells are more difficult to transfect the first passage or two out of the freezer. Thereafter, with some cell lines the transfection efficacy remains relatively constant for over 100 passages. With other cell lines, transfection efficacy may either increase or decrease with passage.

Cell Number (Grade of Confluency)
Cell lines divide exponentially when there is space on the substrate (tissue culture dish). The growth rate is negatively affected by cell density (contact inhibition), depletion of nutrients, and metabolic end products (e.g., pH). The extent of reporter gene expression is directly correlated with the cell number at the beginning of transfection and the growth rate prior to cell lysis.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. General Notes on Primer Design in PCR*
2. Real-Time PCR: General Considerations, Rev A
3. Keys to Successful Densitometry
4. Successful PCR amplification and subcloning of a GC-rich DNA fragment
5. Designing a Successful qRT-PCR Experiment
6. Precursor miRNAs for Successful miRNA Functional Studies
7. Recommendations for Successful siRNA Library Screens
8. Setting up Successful siRNA Library Screens
9. Successful stabilization of gene expression profiles
10. Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell Line
11. Optimizing Transfection Conditions for Studying Signal Transduction Pathways
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... ... June 27, 2016 , ... Newly created ... and solutions to the healthcare market. The company's primary focus is on new ... and marketing strategies that are necessary to help companies efficiently bring their products ...
(Date:6/24/2016)... DIEGO , June 24, 2016 ... more sensitively detects cancers susceptible to PARP inhibitors ... circulating tumor cells (CTCs). The new test has ... HRD-targeted therapeutics in multiple cancer types. ... targeting DNA damage response pathways, including PARP, ATM, ...
(Date:6/23/2016)...   Boston Biomedical , an industry leader ... target cancer stemness pathways, announced that its lead ... Designation from the U.S. Food and Drug Administration ... gastroesophageal junction (GEJ) cancer. Napabucasin is an orally ... stemness pathways by targeting STAT3, and is currently ...
(Date:6/23/2016)...  The Prostate Cancer Foundation (PCF) is pleased to announce 24 ... for prostate cancer. Members of the Class of 2016 were selected from a ... Read More About the Class of 2016 PCF Young Investigators ... ... ...
Breaking Biology Technology:
(Date:6/9/2016)... June 9, 2016 Paris ... Teleste,s video security solution to ensure the safety of people ... during the major tournament Teleste, an international ... and services, announced today that its video security solution will ... to back up public safety across the country. The system ...
(Date:6/2/2016)... -- Perimeter Surveillance & Detection Systems, Biometrics ... Support & Other Service  The latest report ... analysis of the global Border Security market . ... $17.98 billion in 2016. Now: In November ... software and hardware technologies for advanced video surveillance. ...
(Date:5/12/2016)... WearablesResearch.com , a brand of Troubadour Research & ... Q1 wave of its quarterly wearables survey. A particular ... a program where they would receive discounts for sharing ... "We were surprised to see that so many ... CEO of Troubadour Research, "primarily because there are segments ...
Breaking Biology News(10 mins):