I want to thank those people who responded to my query about getting PBMC from cyno blood. There were a number of different suggested methodologies (and lots of commiseration about the difficulties!). We are going to try a few of the methods suggested below and compare them; anyone who is interested in results should email me in no less than about 6-8 weeks. The responses are listed below, with editing only to remove the names of the innocent. (PS, I wish to reiterate the suggestion that all procedures involving centrifugation be provided in terms of force (number of g's); RPM is only relevant if you are using exactly the same bucket and centrifuge--and so if you don't provide that information, becomes almost meaningless!) mr This is what I have learned from others: They dilute the ficoll gradient with RPMI (80 - 90 % Ficoll and 20 - 10% RPMI). Sometimes, it even seams to vary between animals. If I would have to do that I would make a few diluted ficoll gradients with a density between 1.077 and 1.070 (density of ficoll is 1.077) and would try them \ compare them. 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. The only thing I've found that works consistently for cyno blood is a 53% Percoll gradient. I have had bad results with Ficoll gradients as well as the BD CPT tubes. Having worked with baboon, rhesus, and cynomolgous blood I have to say that for some reason cyno blood alone out of the three is very difficult to get good pbmc out of. For some reasons cynos just don't work well with ficoll separation. we've tried altering ficoll spin time and speed to no avail. Nothing that we tried worked consistently, most of the times the layers are just plain nasty. But for the record we dilute 15 mo of blood in 15 ml of RPMI 1640, then overlay over 15ml of room temp Ficoll-paque (Pharmacia). Centrifuge for 30 minutes at 1450, room temp and brake off. Pull of PBMC layer (usually down lower than you would expect) into a 50ml centrifuge tube and fill tube with RPPMI 1640. Centrifuge at 1500 for 10 minutes, decant supernatant and resuspent pellet in 10 ml RPMI. Due to the usually poor preps we then lyse the red blood cells in the pellet by addign 20 or so ml of ammonium chloride (stem cell technologies), incubate on ice for 10 minutes, fill tube with RPMi and centrifuge again. Decant supernatant and resuspend clean pellet and perform cell count. One of my colleagues claimed that she got better results by spinning the ficoll at 2000 instead of 1450, but again this was not consistent from one cyno to another. We are using the standard ficoll procedure but first we dilute the ficoll to 90% with water. Using this technique we get good separation and good yields. I'm afraid there may not be a good solution for you. During my work, I had dozens of cyno samples every few days to monitor the immune status of the transplant recipients. I guess my question to you would be what are you doing with the samples? I found that a simple RBC lysis (ammonium chloride) was sufficient for the samples that only needed routine flow analysis. But for everything else, sorting antigen reactive B cells, culture assays and the such, I HAD to use a gradient (and buy another centrifufge!). In the beginning I tried ALL of the separation medias out there. LSM produced by Mediatech http://www.cellgro.com/catalog2/results2.cfm?ID=75 ) worked significantly better than ANY other product. Trust me on this one, for cyno blood its the best. We still use Ficol gradients to isolate PBMC from ACD blood. We routinely recover 2 million cells per mL blood. We have used this procedure monthly for the past 7 years with cynomolgus macaques. It is time consuming and not without problems (resulting in loss of sample). We routinely isolate PBMC's from cyno's as follows: Dilute heparinized blood approx 1:4 with PBS Layer onto 90% Ficoll-Paque (diluted with PBS) Centrifuge 30minutes at 1400rpm (~400g) without the brake. >From this point, it's fairly routine and the same as human PBMC's. We've also had some luck using the CPT vacutainer tubes from BD. Fresh blood works best with this method. Monkey red blood cells are not easily eliminated when using regular Ficoll-Paque however, this problem is circumvented by adding an anti-red blood cell MAb to the blood before centrifugation on Ficoll. The PBMC were usually cleared from RBC although not all the times. The buffy coat from 5 mL of blood is diluted to a final volume of 2 mL with RPMI. An aliquot of 50 µL of Red-Out (Robbins Scientific Corp., Sunnyvale, CA), a murine monoclonal antibody to human red blood cells, is added and the suspension is incubated for 5 min. The suspension is diluted to 8 mL final with RPMI and overlayed on 5 mL of Ficoll-Paque (Pharmacia). The preparation is spun at 2200 rpm (Sorvall GLC-2B, Dupont) for 30 min. without brake. The PBMC layer is harvested and washed twice in 10 mL of RPMI at 300g for 10 min.
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