cell blebbing with flou4-AM
kavitha thangaraj
Friday, 13 March 2009 06:18 UTC
Im Doing my Phd. currently working on live cell imaging.
I’m using fluo4-am (ca indicator, final concentration 5micromolar)it is dissolved in DMSO. As soon as i add the fluo4 to the cells they started blebbing (necrosis?).
i tried with DMSO alone. the cells (L6, skeletal myoblast) look quite good with DMSO.they are blebbing only with fluo4 dye. I’m also using hoechst dye to stain nucleus. i incubate them for 30 min. de-esterification is for 20 min.
can any one answer for my question?
thanks in advance.
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Hi Cedric&Ruogang
How are u both? Cedric ur suggestions are really working nice. thanks for the input. are these dyes covalently binding to the ca or what ever it is? or it is a dynamic binding?cedric: you can u use either ringer buffer or FBS free medium for loading dyes. especially FBS free media will give u nice fluorescence intensity with low micromolar concentration. can u tell me what kind of dishes u use for imaging and growing cells. if possible can u mail me the pictures of dishes u use. this is my mail ID kavi_kavir@yahoo.co.in.
thanks in advance.
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