STERILE even before filtering, but filtered again as an extra precaution
Mixing container:
for 140 ml and 140 ml, use 250 ml flask.
for 50 ml, use 50 ml blue top flask.
Storage container:
for 140 ml, 250 ml flask labeled "5% - high prog" and 50 ml flask labeled "10% - high prog",
for 100 ml, 250 ml flask labeled "5% 1 KyMg low prog".
for 50 ml, 50 ml blue top flask labeled "5% 1 KyMg low prog".
| INGREDIENT | 170 ml 5% - high prog |
140 ml 5% - high prog |
100 ml 5% 1 KyMg low prog |
80 ml 5% 1 KyMg low prog |
50 ml 5% 1 KyMg low prog |
|
| BME from Sigma | 154.8 ml | 127.5 ml | 79.0 ml | 63.2 ml | 39.5 ml | |
| Serum | 8.5 ml (plus 2 ml for 10%) |
7.0 ml (plus 2 ml for 10%) |
5.0 ml | 4.0 ml | 2.5 ml | |
| Usual additive mixture | 5.95 ml | 4.9 ml | 3.5 ml | 2.8 ml | 1.75 ml | |
| 10 Ky 100 Mg | none | none | 10.0 ml | 8.0 ml | 5.0 ml | |
| mod SVM | 0.77 ml | 0.63 ml | 0.45 ml | 0.36 ml | 0.22 ml | |
| Progesterone 100 µM | none | none | 10 µl | 8 µl | 5 µl | |
| Progesterone 1 mM | 17 µl | 14 µl | none | none | none | |
| AraC 150 µM | none | none | 2.0 ml | 1.6 ml | 1.0 ml |
From the 170 or 140 ml of "5% - high prog", all but 40 ml are filtered as prepared, and the remaining 40 ml are filtered after addition of 2 ml of serum. This 42 ml now has 10% serum.
The Sigma BME has Earle's salts and no glutamine; catalog # B 1522. There is sufficient bicarbonate in this medium, as opposed to ICN Flow's BME for which one had to add bicarbonate.
The additives solution contains glutamine, which breaks down at warm temperatures to glutamate, which is toxic to neurons. For that reason, it is important not to let solutions containing glutamine (such as the additives) warm up until necessary later in the procedure. Consequently, while the frozen serum and modified SVM tubes can be warmed in the 37 ºC water bath, the additives should be brought to room temperature in a beaker containing unheated
de-ionized water.
The modified Stable Vitamin Mix is a slurry, so it must be mixed immediately before taking some up in the pipette. Mix by taking slurry up in the pipette and then expelling it, then take SVM from the middle height of the fluid to get an average mixture.
Filter with a pre-washed 0.2 µm nylon filter, a syringe filter for volumes of 100 ml or less, and a stand filter for larger volumes.
Copyright © 2002 Dr. Michael M. Segal, Department of Neurosurgery, Brigham & Women's Hospital, Boston MA 02115, USA. Many of the solutions are based on recipes from the Furshpan and Potter labs; the recipes given here are the versions we use now in the Segal lab. Individual copies may be made of this page for use in educational institutions.