Hello everyone, I'm trying to obtain good mitotic spreads for FISH application. Because I'm a beginner, I have some question I cannot resolve on web research. So I hope someone, with his/her experience could help me. I work on immortalized human fibroblast line. For my first experience I worked in this way: 450 thousand cells for plate; after 2 day add thymidine and leave for 16hours. Next day after 4 hours for release of thymidine add colcemid 0.2ug/ul and leave for 30-60-90 min. at 37°C. Collect then medium, PBS and trypsinize cells. Then centrifuge 200g x 3-5' and add 0.075M KCl drop by drop. Leave 15 min at 37°C and after add 60ul of cold fresh prepared fixative (methanol:acetic acid 3:1), inverting tube several time before to centrifuge at 800rpm for 8 min. (I know I have to talk in gravity but my protocol talk about rpm... without talk of rotor, but from the video I saw it appears the same i used). After centrifugation resuspend in 1ml of cold fixative drop by drop and transfer in eppendorf, inverting tube several time. Centrifuge 500rpm (same discussion of above) 5'. Repeat 3 times these last steps. At the last I prepared spreads in this way: starting with ethanol cleaned slides I wash in milliQ water. Then on every slide I added drops of 15 or 20ul of resuspended fixative:cells solution from about an height of 15-20cm. Then i let evaporate on flame for 10 seconds and leave slides overnight on bench. Next day stain with Giemsa and see. What i saw is a good number of nuclei but for every slide i saw 5-6 mitotic spreads of medium-good quality. So i could say that for 15-20 nuclei I have 1 spread with chromosomes... Is this a good rapport or percentage? Nuclear membrane of cells appears in any case as fragmentated and i can see chromosomes insides.... Is a good number of spreads for nuclei? If not, can you give me an hint to increase number of spreads in contrast of nuclei?
If you want answer to [email protected] Thanks a lot. Bye bye Pasquale _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
