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problem with cell cycle analysis


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#1 deep

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Posted 28 June 2010 - 08:50 PM

Hello everybody,
I am novice to cell cycle anaysis and done this procedure ( Propidium iodide staining) for the first time in A431 cancer cell lines.
my results were discouraging ;)
the person who analysed my samples for cell cycle analysis told me that the cells are beyond their duplicate forms . what does it mean? (multinucleated cells?)
also she had to lower the voltage to analyse histograms. what it suggests?
Please help me to solve my doubt and how can I overcome these problems?
method I followed-
- seed 10*5 to 10*6 cell in 50 to 80mm petri dish
-after overnight incubation I added drug to petri dishes
after 48 hrs I fixed the cell in ethanol(stored at -20 deg celcius)
- kept cells in 4 deg celcius.
next day did PI staining.

thank you..
deep

#2 josyng

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Posted 28 June 2010 - 10:48 PM

Hi Deep
the cells u r using hav the property of forming clumps very quickly. So when u r acquisitioning the sample by flow.. vortex properly before.. and in between also... maximum u can use 1 million cells for staining.. and after fixation with ethanol .. store at -20c for overnight to 48 hrs will help in better results...
rgds
Jo

#3 deep

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Posted 29 June 2010 - 08:25 PM

Thanx Jo for your sugestions.

#4 zodiac1505

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Posted 01 July 2010 - 06:53 AM

Do you have the possibility to stain and look at the cells the same day as harvesting? Also when you fix the cells try adding the ethanol(ice cold, 70%,3ml) drop by drop while vortexing the cells at a low speed setting. Leave the cells on ice for 30mnts, wash once with PBS and stain with PI for 30 mnts (RT, dark). This protocol has worked for me well when I do cell cycle analysis but I do the FACS immediately after.




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