Sirus red

a.vijayan ort020 at nof.abdn.ac.uk
Sat Apr 8 05:52:50 EST 1995


caro (csaucier at pharma1.pharma.mcgill.ca) wrote:
: Hi!

: 	I'am looking for a protocol for measuring the level of collagen releases
: by cells in the culture medium by sirius red staining. 

: Thank you very much
: Caroline 
 
Interesting!!!

I have used this assay once.
I give you the appropriate reference and a short protocol.
Ref: Anderson,S.M.L and Elliott, R.J(1991) Biochemical Society transactions, vol
19,389S. Evaluation of a new rapid collagen assay.
This assays is known as Sircol Collagen Assay.
A kit for this assay is available from Qubis Ltd., Belfast,BT9 5BN Northern
Ireland. Tel: +44(0) 232 2333699 Fax: +44(0)232 233699.
Protocol:
1.Reagent blanks(100ul of 0.5M acetic acid)
2.Collagen standards(12.5, 25.0, 50.0ug).
3.Test samples,(volumes:10 to 100ul)
Adjust all tube content to 100ul with 0.5M acetic acid 
To each tube add 1ml Sircol dye reagent and cap.

Shake gently for 30 mins
Centrifuge in a microcentrifuge at >5000g for 5 mins
Drain the unbound dye by inverting the tubes and remove droplets with a piece of
tissue
To each tube add 1ml of alkali reagent(0.5M NaOH) and mix on vortex mixer.
Set Spectrophotometer to 540nm and measure abs within 3hrs.
I have done this on microtitre 96 well plates using a plate reader.
I hope this helps and hoping to hear the outcome.
Regards,
a.vijayan at aberdeen.ac.uk



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