Mario, We have been isolating cyno PBMC for many years using Lymphoprep gradient from Nycomed. It works quite well. First we dilute the heparinized blood 1:3 with media or PBS. We then layer it over the Lymphoprep at a ratio of 1 part Lymphoprep to 2 parts diluted blood. Spin for 30 minutes, room temp, no brake at a speed of about 1800 RPM. Harvest the interface, which is sometimes a little more diffuse than you would see in a human prep but easily retrievable. We wash twice and lyse red blood cells if necessary and the cells are ready to use. We routinely get about 10 million cells from 5 ml of blood. They are very viable and are essentially all mononuclear cells. We spin one of the washes at about 1200 RPM. This helps to get rid of most of the platelets that sneak in when we harvest our interface. Some animals will have more red cell contamination of the interface than others. It seem that the more you bleed them, the more red cell contamination you see. Naive animals usually have very clean interfaces. It is not really a problem for us because we either just lyse the RBC using standard non-fixative red lysis buffer or we leave them. Often times we are freezing the cells down for later use or using them in culture conditions were a few RBC are ok. This Lymphoprep method is easy and not very time consuming. We can harvest PBMC from a dozen or more monkeys in a morning and that includes bleeding them! Hope this helps. Andrea Bree Wyeth/Genetic Institute >>> Mario Roederer <roederer@drmr.com> 9/27/01 11:14:13 AM >>> We are trying to find an easier way to isolate PBMC from cyno blood than a continuous gradient. For those of you using cells from Cyno's, can you send me your PBMC isolation protocol? thanks mr
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