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:5/23/2017)... ... 2017 , ... Kathy Goin is joining myClin ... years of expertise in establishing and leading clinical operations at Sponsors including Trevena, ... therapist, through a variety of leadership roles in Clinical Operations, to her current ...
(Date:5/23/2017)... ... 2017 , ... Cambridge Semantics , the leading provider of Big Data ... Conference and Expo in Boston May 23-25 with a featured speaker and solution ... Data Lake is also a finalist for the Best of Show award. , James ...
(Date:5/22/2017)... ... ... Stratevi, a boutique firm that partners with healthcare companies to creatively develop and ... downtown Boston at 745 Atlantic Ave. , “We are seeing that even the ... value they provide, not just to patients, but also payers. Having a presence in ...
(Date:5/22/2017)... ... May 22, 2017 , ... ... it is exhibiting in booth B2 at the Association for Pathology Informatics ... , In addition to demonstrating its Cancer Diagnostic Cockpit and Consultation Portal, ...
Breaking Biology Technology:
(Date:3/27/2017)... , March 27, 2017  Catholic Health ... and Management Systems Society (HIMSS) Analytics for achieving ... Adoption Model sm . In addition, CHS previously ... U.S. hospitals using an electronic medical record (EMR). ... its high level of EMR usage in an ...
(Date:3/22/2017)... , March 21, 2017   Neurotechnology , ... recognition technologies, today announced the release of the ... which provides improved facial recognition using up to ... a single computer. The new version uses deep ... accuracy, and it utilizes a Graphing Processing Unit ...
(Date:3/16/2017)... 2017 - Against identity fraud with DERMALOG solutions "Made in Germany "  ... ... project, multi-biometric solutions provide a crucial contribution against identity fraud. (PRNewsFoto/Dermalog Identification Systems) ... Used combined in one project, multi-biometric solutions provide a crucial contribution against ... ...
Breaking Biology News(10 mins):