Dear Flowers, A colleague of me is requested to develop a method to measure the DNA contents of rat hepatocytes possibly in combination with BrdU incorporation. The method we are trying to develop includes the following steps: -in vivo BrdU labelling -isolation of rat hepatocytes from a piece of the liver (so I think perfusion is not possible) -fixation with ethanol 70% -intra nuclear staining of BrdU using anti-BrdU FITC using HCl denaturation and Triton X-100 -subsequent DNA staining with PI -flow cytometric assessment An additional complicating factor is that we need a method which makes it feasible to handle some 50 samples a day. Until now some experiments have been performed with cells isolated by mechanical disruption (200 µm mesh filter) and some experiments to demonstrate the presence of BrdU. With respect to the obtained DNA profiles it appears that the isolation method has not been very successful because we get broad (twin) peaks and also G0/G1 versus G2/M ratio's which are not in accordance with the literature. The tuning of the FCM seemed ok because we did get better results when measuring spleen cells. Concerning the BrdU measurements we got a high background staining which may be indicative for a sub-optimal isolation method. So my question is: does anyone have experience with this application? If so would you please contact me either by phone, fax or e-mail? Or if you know someone who has experience with this could you give his or her address? With kind regards, Joost Bruijntjes
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