Navigation Links
Primary cells, rat heart muscle

Multiporator Transfection Protocol Protocol No. 4308 915.035 12/1999 Cell line Primary cells: Adult heart muscle cells from rat, 6 days in culture Transfection with Plasmid pCMV--galactosidase (in bidistilled H2O) Electroporation buffer Eppendorf Isoosmolar Electroporation Buffer (PI) Culture medium M199 / HEPES (2 mM carnithin, 5 mM taurine, 10 M cytosine-arabinofur.)+ 20% FCS Outgrowth medium HEPES-buffer (140 mM NaCl, 3.6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 11 mM glucose, 100 U/ml penicillin, 100 g/ml streptomycin, 20 mM HEPES, pH 7.4) without phenol red + 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Dr. Gerhild Taimor Justus-Liebig-Universitt Physiologisches Institut Aulweg 129 D-35392 Gieen
Phone +49 641 9947243 Fax +49 641 9947239 mail: gerhild.taimor@physiologie.med.uni-giessen.de
  1. Harvest the cells by collagenase treatment (0.4 mg/ml, 30 min., 37 C) and centrifuge them (for 2 minutes, 300 x g, at room temperature).
  2. Resuspend the cells in M199 / 0.5% FCS, determine the number of cells and centrifuge them (for 2 minute s, 300 x g, at room temperature). Remove supernatant.

    Note: The overall incubation time in the Eppendorf Electroporation Buffer must not exceed 30 minutes to guarantee a successful electroporation!

  3. Resuspend the cells in Isoosmolar Electroporation Buffer. When doing so, set the cell concentration to 1 x 104 - 105 cells/ml.
  4. Add and mix plasmid DNA (5-20 g/ml final concentration, in bidistilled H2O).
  5. Transfer 400 l cell suspension into electroporation cuvettes (2 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 100 V Time constant (T) 50 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 3-5 ml HEPES medium without phenol red / 20% FCS, and cultivate them in a 35 mm culture dish.

Detection methods for transfection:

The expression of the plasmid pCMV--galactosidase can be detected clearly after 24-48 hours with X-gal-staining under a microscope.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Human Primary Preadipocytes and Differentiated Adipocytes
2. Primary cells, rat heart endothel
3. Efficient Delivery of siRNAs to Human Primary Cells: Electroporation vs. Chemical Transfection
4. Deliver siRNAs Into Primary Cells
5. High Throughput siRNA Delivery In Vitro: From Cell Lines to Primary Cells
6. Electroporation of Primary Bone Marrow Cells
7. Efficient Primary Structure Elucidation of Disulfide-bridged Peptides
8. Embryonic stem cells, mouse
9. Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/11/2017)... ... 2017 , ... At its national board meeting in North ... the co-founder, CEO and chief research scientist of Minnesota-based Advanced Space Technology and ... ARCS Alumni Hall of Fame . ASTER Labs is a technology development ...
(Date:10/11/2017)... -- VMS BioMarketing, a leading provider of patient support solutions, has ... (CNE) network, which will launch this week. The VMS CNEs ... professionals to enhance the patient care experience by delivering peer-to-peer ... care professionals to help women who have been diagnosed and ... ...
(Date:10/10/2017)... , ... October 10, 2017 , ... San Diego-based team ... its corporate rebranding initiative announced today. The bold new look is part of ... the company moves into a significant growth period. , It will also expand its ...
(Date:10/10/2017)... , Oct. 10, 2017 SomaGenics announced the ... NIH to develop RealSeq®-SC (Single Cell), expected to be ... small RNAs (including microRNAs) from single cells using NGS ... the need to accelerate development of approaches to analyze ... "New techniques for measuring levels of mRNAs ...
Breaking Biology Technology:
(Date:4/3/2017)... WASHINGTON , April 3, 2017 /PRNewswire-USNewswire/ ... single-cell precision engineering platform, detected a statistically ... cell product prior to treatment and objective ... highlight the potential to predict whether cancer ... prior to treatment, as well as to ...
(Date:3/30/2017)... -- The research team of The Hong Kong Polytechnic ... by adopting ground breaking 3D fingerprint minutiae recovery and matching technology, ... accuracy for use in identification, crime investigation, immigration control, security of ... ... A research team led by Dr ...
(Date:3/29/2017)... 29, 2017  higi, the health IT company that ... North America , today announced a Series B ... of EveryMove. The new investment and acquisition accelerates higi,s ... to transform population health activities through the collection and ... higi collects and secures data today on behalf ...
Breaking Biology News(10 mins):