Collagen gels not setting!! - (Jan/20/2005 )
I am trying to make solid collagen gels to release from the plates as rafts. I am currently making them up as follows: 100ul MEM, 900ul diluted collagen, 20ul NaOH and 2ul bicarbonate.
Does anyone have any suggestions as to what i can do? If anyone has a protocol they know works i would be grateful if you could post it for me.
I have done a lot of work with making collagen gels. I would check your collagen concentration and possibly increase it. Also, check your end pH; if it is too low then you will not get proper gelling.
I use 8 parts collagen, 1 part reconstitution buffer, and 1 part 10x media. The reconstitution buffer consists of HEPES and sodium bicarbonate at the concentration in the 1x media plus 0.2N final conc. NaOH (in the buffer). Others have used down to 0.05N NaOH, but you have to see what works best for you. My final concentration of collagen is about 2.8 mg/ml, which seems to work well for growth of cells.
Please email me at email@example.com if you have further questions.
To clarify the procedure for making collagen gels, the procedure was based on two articles by Ma et al (1997) and Nakashiro et al (2000). These are referenced in my research article published in the journal, Prostate (Barrett et al 2005). I made collagen gel mixes using rat tail collagen type I at an initial (stock) concentration of about 3.5 to 5 mg/ml in 0.02N acetic acid, which was diluted to a final concentration (for cell growth) of about 2.6 to 2.8 mg/ml using 10X media and a 10X concentration of a reconstitution buffer (this buffer is specified in the Ma and Nakashiro references, but I made a variation of the buffer to improve collagen gelling and cell growth for my conditions). It is important to make sure the stock reconstitution buffer contains the appropriate 10X concentration of components (especially HEPES and sodium bicarbonate) for the media in which the cells will be grown. The concentration of NaOH in the 10X reconstitution buffer should also be considered based on final concentration of collagen and the type of cells you plan to grown in collagen. I used a 0.2N NaOH conc. in the 10X reconstitution buffer, but the papers by Ma and Nakashiro noted the use of 0.05N NaOH. It would be good to test what would work best for your conditions.
I hope this helps clarify the procedure. I have a new email address, which is firstname.lastname@example.org. Feel free to contact me directly for further help with setting up this procedure, troubleshooting issues, and downstream applications.