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:2/9/2016)... ... February 09, 2016 , ... With a presidential election in November and the ... will bring together over 500 top healthcare leaders for a night and day of ... organized by MBA students of the University of Pennsylvania’s Wharton School, will be held ...
(Date:2/9/2016)... ... February 09, 2016 , ... Tunnell Consulting, Inc. announced that Frédéric Lefebvre ... will focus on acquiring new accounts and work closely with existing Tunnell clients throughout ... brings to our European clients more than 15 years of experience in the pharmaceutical ...
(Date:2/9/2016)... YORK , Feb. 9, 2016 This ... analyzes the current and future prospects of the market ... this report include companies engaged in the manufacture of ... an executive summary with a market snapshot providing the ... scope of this report. This section also provides the ...
(Date:2/8/2016)... /PRNewswire/ - BIOREM Inc. (TSXV: BRM) ("Biorem" or "the Company") ... clean technology companies in the TSX Venture 50 TM . ... on the TSX Venture Exchange, in each of five major ... & life sciences, diversified industries and technology – based ... on investment, market cap growth, trading volume and analyst coverage. ...
Breaking Biology Technology:
(Date:1/25/2016)... , Jan. 25, 2016  Glencoe Software, the world-leading ... and publication industries, will provide the data management solution ... (NPSC). ... Phenotypic analysis measures the ... organisms, allowing comparisons between states such as health and ...
(Date:1/21/2016)... --> ... report "Emotion Detection and Recognition Market by Technology (Bio-Sensors, NLP, ... Voice Recognition and Others), Services, Application Areas, End ... published by MarketsandMarkets, the global Emotion Detection and ... Billion by 2020, at a CAGR of 31.9%, ...
(Date:1/20/2016)... SAN JOSE, Calif. , Jan. 20, 2016 ... leading developer of human interface solutions, today announced ... touch controller solution for wearables and small screen ... appliances such as printers. Supporting round and rectangular ... the S1423 offers excellent performance with moisture on ...
Breaking Biology News(10 mins):