Hi, I want to use elastase to optimize the dissociation of cells from spheroids. Currently I am screening different enzymes and combinations of enzymes for that purpose.
However, for elastase I found very little literature on what concentration (mg/ml) is usually used during the dissociation of different tissues.
It would be even better to state the activity (U/ml) but this is even more rare.
If you are familiar with the use of elastase I would also appreciate information on how to prepare a proper stock solution (concentration, buffer, pH ...) since there is also differing information on that on the web.
Thanks a lot!
Dear Dominik Egger!
Here is the answer. Please check it!
Sincerely yours!
Dr. Bratko Filipič
Email: [email protected]
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MATERIALS AND METHODS
Male guinea pigs weighing about 100 g were used for the study. They were killed by decapitation, and the lungs were quickly removed, blotted on filter paper, weighed, and placed in 10 ml of the solution to be tested for cell-dis- persing activity. In some experiments kidney tis- sue was used, in which case the capsule and calyces were removed before weighing. A calcium-free salt solution devised by Marcus, Cieciura, and Puck (6) was used to suspend enzymes or other agents in these experiments. This solution contains NaC1, KC1, glucose, NaHCO~, and Trypsin 1: 300. Trypsin 1:300 was omitted from the solution when other agents were being tested. The enzyme preparations used in this study were: Trypsin 1:300, General Biochemical Co. (a crude pancreatic trypsin) ; trypsin, Sigma (2 • crystallized); a-chymotrypsin, Boehringer- Mannheim Co. (crystallized) ; Collagenase, Type I (contains protease and peptidase activity), Sigma; Collagenase (contains protease and peptidase activity), Nutritional Biochemical Co.; Protease, Type V (purified), Sigma; Elastase (crystallized), Cudahy Laboratories.
Cell suspensions were prepared quantitatively so that results could be expressed in terms of the number of cells freed from a milligram of tissue. The tissue was placed in 10 ml of test solution in a 50-ml round bottom centrifuge tube and chopped with scissors for exactly 1 min. The lung tissue was allowed to float (settle in the case of kidney) for 30 sec, and the fluid, which contained mostly red blood cells, was siphoned from the bottom of the tube.
Test solution (10 ml) was added to the tissue, and the process of chopping and washing the tissue was repeated four more times. The chopped tissue and 100 ml of test solution were stirred in a "trypsinization flask" on a magnetic stirrer for 30 min. Cell suspension (25 ml) was pipetted from the flask, and cells were collected by centrifugation at 2000 • g for 20 min. Fresh test solution (25 ml) was added to the trypsinization flask, and stirring was continued for 30 min more. The process of stirring the tissue and collecting aliquots of the cell suspension at 30-min intervals was re- peated two more times. Aliquots were pooled. Cells were suspended in Hanks' solution with a Pasteur pipet and passed through one layer of cheesecloth to remove fibers and pieces of tissue. Red blood corpuscles were removed by centri- fuging the suspension on a gradient of 1 ml of 23% albumin in a Goetz tube at 2000 • g for 20' min. The cells in the supernatant fluid were col- lected and counted on a hemacytometer. Values are expressed as the number of cells per milligram of original tissue.
Thanks for the answer. However, what was the used concentration/activity in the buffer?
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