Navigation Links
HL-60

Multiporator Transfection Protocol Protocol No. 4308 915.028 11/1999 Cell line HL-60, human monocyte like cells (suspension cell line) Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI 1640 / 10% FCS Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Hans Hcker Institut fr Med. Mikrobiologie, Immunologie und Hygiene
Technische Universitt Mnchen Trogerstr. 4a D-81675 Mnchen
Phone +49 89 4140 4184 Fax +49 89 4140 4183 e-mail: hans.haecker@lrz.tu-muenchen.de
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 200 x g, at room temperature).
  2. Resuspend the cells in RPMI 1640 / 0.5% FCS, determine the number of cells and centrifuge them (for 5-10 minutes, 200 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 Hypoosmolar Electro poration Buffer. When doing so. set the cell concentration to 2.5 x 106 cells/ml.
  4. Add and mix plasmid DNA (25 g/ml final concentration, in bidistilled H2O).
  5. Transfer 800 l cell suspension into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 760 V Time constant (T) 60 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 3-5 ml RPMI 1640 / 10% FCS, and cultivate it in a 35 mm culture dish.
Detection methods for transfection:
The expression of the plasmid pEGFP-N1 can be detected clearly after 24-48 hours with the aid of FACS analysis or under a fluorescence microscope. Result: Survival rate: 80% Transfection rate: 44% based on the number of surviving cells
35% based on the initial number of cells used for the experiment Results were measured 24 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Primary cells, rat heart endothel
2. Primary cells, rat heart muscle
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... 27, 2016 /PRNewswire/ - BIOREM Inc. (TSX-V: BRM) ("Biorem" or ... its major shareholders, Clean Technology Fund I, LP and ... based venture capital funds which together hold ... a fully diluted, as converted basis), that they have ... entire equity holdings in Biorem to TUS Holdings Co. ...
(Date:6/27/2016)... ... 2016 , ... Parallel 6 , the leading software as a service ... Virtual Patient Encounter CONSULT module which enables both audio and video telemedicine communication ... , Using the CONSULT module, patients and physicians can schedule a face to face ...
(Date:6/27/2016)...  Liquid Biotech USA , ... Sponsored Research Agreement with The University of Pennsylvania ... cancer patients.  The funding will be used to ... clinical outcomes in cancer patients undergoing a variety ... employed to support the design of a therapeutic, ...
(Date:6/24/2016)... 24, 2016 Epic Sciences unveiled a ... susceptible to PARP inhibitors by targeting homologous recombination ... The new test has already been incorporated into ... cancer types. Over 230 clinical trials ... pathways, including PARP, ATM, ATR, DNA-PK and WEE-1. ...
Breaking Biology Technology:
(Date:5/12/2016)... , May 12, 2016 WearablesResearch.com ... just published the overview results from the Q1 wave ... the recent wave was consumers, receptivity to a program ... data with a health insurance company. "We ... to share," says Michael LaColla , CEO of ...
(Date:5/3/2016)... 3, 2016  Neurotechnology, a provider of high-precision ... Automated Biometric Identification System (ABIS) , a complete ... MegaMatcher ABIS can process multiple complex biometric transactions ... of fingerprint, face or iris biometrics. It leverages ... and MegaMatcher Accelerator , which have been ...
(Date:4/28/2016)... -- First quarter 2016:   , Revenues ... first quarter of 2015 The gross margin was 49% ... and the operating margin was 40% (-13) Earnings per ... from operations was SEK 249.9 M (21.2) , Outlook ... 7,000-8,500 M. The operating margin for 2016 is estimated ...
Breaking Biology News(10 mins):