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:12/8/2016)... ... December 08, 2016 , ... Lajollacooks4u, San Diego’s premier team building ... team building events, new program offerings and company expansion. , This is ... to include groups of over 30 people. Ever since, Lajollacooks4u has seen significant demand ...
(Date:12/8/2016)... -- OncoSec Medical Incorporated ("OncoSec") (NASDAQ: ONCS ... today announced financial results for the fiscal first ... on our commitment to address an unmet medical ... pleased with the early clinical response data presented ... focused on advancing our lead program – ImmunoPulse® ...
(Date:12/8/2016)... ... 2016 , ... From wearable devices that can improve performance ... Thursday, December 15th a panel of entrepreneurs, innovators and a Philadelphia Eagles front ... Smart Talk session. Smart Talk will run from 8:30 – 10:30 a.m. at ...
(Date:12/8/2016)... 2016 AskLinkerReports.com has published a report ... Amyloglucosidase Industry 2016 Market Research Report. From a basic outline ... overview are all covered in the report. This report projects ... analysis of the Amyloglucosidase industry. ... , , ...
Breaking Biology Technology:
(Date:12/8/2016)... Research Future published a half cooked research report on Mobile Biometric ... Market is expected to grow over the CAGR of ~35% during ... ... Mobile Biometric Security and Service Market is increasing at a ... security from unwanted cyber threats. The increasing use of mobile device ...
(Date:12/8/2016)... 2016  Singulex, Inc., the leader in Next Generation ... a license and supply agreement with Thermo Fisher Scientific, ... Singulex access to Thermo Scientific BRAHMS PCT (Procalcitonin), a ... used to diagnose systemic bacterial infection and sepsis and ... aid in assessing the risk of critically ill patients ...
(Date:12/7/2016)... 2016 According to a new market research report "Emotion ... Expression, Voice Recognition), Service, Application Area, End User, And Region - Global Forecast ... USD 6.72 Billion in 2016 to USD 36.07 Billion by 2021, at a ... Reading ... MarketsandMarkets Logo ...
Breaking Biology News(10 mins):