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:3/23/2017)... ... 2017 , ... AxioMed president, Jake Lubinski, describes the AxioMed ... deformed, which is identical to how the human discs work to distribute force. ... to its natural state along a hysteresis curve, exactly like a healthy human ...
(Date:3/23/2017)... DUBLIN , Mar 23, 2017 ... "Biorefinery Products: Global Markets" report to their offering. ... The ... from $466.6 Billion in 2016 at a CAGR of 8.9%, ... of energetic and non-energetic bioproducts into seven major product segments: ...
(Date:3/23/2017)... , March 23, 2017  SeraCare Life ... global in vitro diagnostics manufacturers and clinical ... industry,s first multiplexed Inherited Cancer reference ... by next-generation sequencing (NGS). The Seraseq™ Inherited ... with input from industry experts to validate ...
(Date:3/23/2017)... Calif., March 23, 2017  BioPharmX Corporation (NYSE ... for the dermatology market, today reported financial results ... 2017, and will provide an update on the ... year. "We are pleased to report ... for BioPharmX," said President Anja Krammer. "We achieved ...
Breaking Biology Technology:
(Date:3/24/2017)... March 24, 2017 The Controller General of Immigration ... Abdulla Algeen have received the prestigious international IAIR Award for ... Continue Reading ... ... Deputy Controller Abdulla Algeen (small picture on the right) have received the ...
(Date:3/22/2017)... , March 21, 2017 ... Cloud used by retailers such as 1-800-Flowers and ... platform — Product Recommendations and Replenishment. Using Optimove,s machine ... more personalized product and replenishment recommendations to their ... also on predictions of customer intent drawn from ...
(Date:3/13/2017)... Future of security: Biometric Face Matching software  ... ... Matching enables to match face pictures against each other or against large databases. ... Systems) ... the fastest software for biometric Face Matching on the market. The speed is ...
Breaking Biology News(10 mins):