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:9/19/2017)... ... September 19, 2017 , ... ... announced a partnership with Cytena GmbH to launch the CloneSelect™ Single-Cell Printer™ in ... analysis to isolate single cells and provide visual documentation of monoclonality for use ...
(Date:9/18/2017)... ... September 18, 2017 , ... Transportable biomass conversion ... and torrefied wood is the topic of a September 27 webinar ... economic viability of transportable biomass conversion facilities for producing biochar, briquettes, and torrefied ...
(Date:9/18/2017)... ... September 18, 2017 , ... ... business process optimization firm for the life sciences and healthcare industries, announces ... 2017 conference. , What: Digital Transformation in Medical Device – The Journey to ...
(Date:9/14/2017)... ... September 14, 2017 , ... ... flexible scientist program (FSP)-- a flexible business approach similar to a full-time ... SSCI’s extensive project-based analytical and solid-state chemistry services and expertise with flexible ...
Breaking Biology Technology:
(Date:6/23/2017)... N.Y. and ITHACA, N.Y. ... ) and Cornell University, a leader in dairy research, ... with bioinformatics designed to help reduce the chances that ... With the onset of this dairy project, Cornell University ... Consortium for Sequencing the Food Supply Chain, a food ...
(Date:5/23/2017)... 2017  Hunova, the first robotic gym for the rehabilitation and functional ... in Genoa, Italy . The first 30 robots will ... USA . The technology was developed and patented at ... IIT spin-off Movendo Technology thanks to a 10 million euro investment from ... click: ...
(Date:5/16/2017)... 2017   Bridge Patient Portal , an ... MD EMR Systems , an electronic medical record ... have established a partnership to build an interface ... GE Centricity™ products, including Centricity Practice Solution (CPS), ... These new integrations will allow healthcare delivery networks ...
Breaking Biology News(10 mins):