1. Place bottles in a 56oC water bath. Make sure the bottles cannot tip over or otherwise become submerged in the water bath. The temperature is critical for complement degredation, so make sure!
2. Wait until the temperature stabilizes at 56oC (after adding the serum to the water bath)
3. Gently swirl the bottles every 10 minutes until 30 minutes has elapsed. Do not allow the serum to incubate more than 30 minutes!
Note: Heat inactivation may change the appearance of the serum.
The Department of Biological sciences of the University of Maryland (UMBC) has a protocol section very useful for cell culture first time users.
They explain extensively all the components of the work area and the equipment mainly used, from how the hood works to the microscope or the incubators. Also, types of cells that grow in culture, preservation and storage of cells, manteinance (media and growth requirements, harvesting,…) and safety considerations.
It is a very useful guide for those who begin using cell cultures and want to have an overview of everything that it involves. Here is the link to UMBC web site: http://userpages.umbc.edu/~jwolf/method5.htm
Sigma-Aldrich publishes in its learning center web site a very complete guide to cell culture techniques.
It goes from the basic techniques to sterile techniques, freeze and thaw cells, culturing of semi-adherent or suspension cell lines, test for bacteria or fungi in the cultures, detection of mycoplasma,…
In summary, an extensive guide for cell culture beginners. Here is the link:
http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/ecacc-handbook/cell-culture-techniques-12.html
Basic Techniques, Aseptic Technique and Good Cell Culture Practice, Ressuscitation of Frozen Cell Lines, Subculture of Adherent Cell Lines, Subculture of Semi-Adherent Cell Lines, Subculture of Suspension Cell Lines, Cell Quantification, Cryopreservation of Cell Lines, Testing for Bacteria and Fungi, Detection of Mycoplasma by Culture, Testing for Mycoplasma by Indirect DNA Stain.
Laemmli is a sample buffer to use in western blot. 6X sample buffer is added to each protein sample and is boiled or heated for 5-10 minutes.
Recipe to prepare 10 ml:
- 1.2gr SDS (sodium dodecyl sulfate)
- 6mg bromophenol blue
- 4.7ml glycerol
- 1.2ml Tris 0.5M pH6.8
- 2.1ml dwater
warm it a little bit and shake it till everything is dissolved. It takes time.
- add 0.93gr DTT.
Wait until it is completely dissolved.
Aliquote and keep frozen at-20ºC.
Some months ago I thought about starting a blog about science. And here it is. The idea behind it is to serve as a meeting site for scientists around the world that are looking for a protocol, searching advise from other scientists, want to be updated on the latest science news,… Everyone is welcome: from PhD students to PI’s I hope all of you will find here interesting information and the entertaintment you are looking for in those long waiting times that usually we all have during experiments.