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mitotic spreads preparation

pasquale pensieri via methods%40net.bio.net (by p.pensieri87 from gmail.com)
Sun Feb 28 05:55:26 EST 2016


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 p.pensieri87 from gmail.com

Thanks a lot.
Bye bye
Pasquale


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