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Scratch/Wound Healing Assay Substrate?


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#1 jshaya

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Posted 06 July 2009 - 08:22 PM

Hey,

I am trying to do a wound/scratch assay using MCF-7 cells and a stablely transfected MCF-7 cell line to see the difference. I have done a lot of research on the protocol, but the thing I am wondering is can cells migrate on plastic or is it imperative that you put down some sort of substrate i.e. fibronectin, collagen, etc. I tried a preliminary test and the cells didn't show any wound closure (there was some as the days progressed due to proliferation). Should I put down fibronectin or some other ECM factor?

Thanks!

#2 LostintheLab

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Posted 06 July 2009 - 10:02 PM

I have done Scratch wounding assays on MCF7 cells grown on normal cell culture plastic with no problems. They are quite slow migraters-24-48hr for wound closure in my experience. Of course you can grow them on different substrates,this will likely change the migration rates- but then that is an extra factor in the wound healing experiment interpretation, so you would need to be sure of your parameters.

Its strange that you didn't see any migration though- how long did you wait before checking the cells?
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#3 jshaya

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Posted 07 July 2009 - 09:39 PM

I have done Scratch wounding assays on MCF7 cells grown on normal cell culture plastic with no problems. They are quite slow migraters-24-48hr for wound closure in my experience. Of course you can grow them on different substrates,this will likely change the migration rates- but then that is an extra factor in the wound healing experiment interpretation, so you would need to be sure of your parameters.

Its strange that you didn't see any migration though- how long did you wait before checking the cells?


I checked them 24 and 48 hours after creating the wound and there was very little closure. What type of media did you use? I only tried the normal media the cells are grown in, but I am planning on trying reduced serum or at least adding some sort of antiproliferative drug to prevent cell proliferation.

#4 LostintheLab

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Posted 07 July 2009 - 10:30 PM

I have done Scratch wounding assays on MCF7 cells grown on normal cell culture plastic with no problems. They are quite slow migraters-24-48hr for wound closure in my experience. Of course you can grow them on different substrates,this will likely change the migration rates- but then that is an extra factor in the wound healing experiment interpretation, so you would need to be sure of your parameters.

Its strange that you didn't see any migration though- how long did you wait before checking the cells?


I checked them 24 and 48 hours after creating the wound and there was very little closure. What type of media did you use? I only tried the normal media the cells are grown in, but I am planning on trying reduced serum or at least adding some sort of antiproliferative drug to prevent cell proliferation.


The MCF7 cells were cultured in DMEM with 10% FCS added. The media had L-glutamine in it, pretty standard media. Also used reduced serum media with similar responses.
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#5 rrahman1

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Posted 05 November 2009 - 08:42 AM

Hey,

I am trying to do a wound/scratch assay for my project on oestrogen signal via Erα or ERβ to modulate cell migration, the project discriptions are as follows:

Steroid hormones, including oestrogens, may influence cell migration during both wound healing and metastasis. Recent evidence suggests that oestrogen modulates cytoskeletal rearrangement and cell migration via a cell membrane ERin endothelial cells1 and skin fibroblasts2. Using specific ER agonists, this project will investigate their effects on an established cell line using the scratch wound assay. Migration will be measured over different time points. Single point assays in the presence of pertussis toxin will also be performed to determine whether changes in migration are modulated via a non-genomic pathway.

my question here is regarding what cell lines are best suited for this study and the quantities of it that may be possible to conduct an scratch/wound assay on this particular task?.

#6 bob1

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Posted 05 November 2009 - 03:13 PM

MCF7 are ER positive - so this will work, the presence of estrogen slows their growth rate, which is why you are supposed to culture them in phenol red (estrogen analogue) free medium.

#7 rrahman1

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Posted 06 November 2009 - 01:16 AM

MCF7 are ER positive - so this will work, the presence of estrogen slows their growth rate, which is why you are supposed to culture them in phenol red (estrogen analogue) free medium.

the aim formy practical is to measure the effects of oestrogen agonists, ER-alpha and ER-beta\on a established cell line using scratch wound assay at diferent points i.e. 0hr, 4hr, 8hr, 16hr, 24hr and 48hr?.

will i be able to measure these affects by applying the test on cell line MCF7? i've researched many wound protocols i am still confused on how to start my methodoloy and the qunatities of substrate i may need.

#8 bob1

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Posted 08 November 2009 - 03:26 PM

I think your time points are a bit short, I would go with 12, 24, 48, 72 hours post treatment.

#9 rrahman1

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Posted 09 November 2009 - 12:48 PM

I think your time points are a bit short, I would go with 12, 24, 48, 72 hours post treatment.


thanx for the reply bob, i have to test on an early time line and a late time line point so fought 0 being early and 48hrs prepferaby the latests. i have decide to use keratinocytes NCTC cell line to determine effect of oestrogen agonist alpha/beta to modulate cell migration. by changing different concnetrations with time intervals. i was wondering the concentration levels to use for ppt er-alpha agonist, dpn er-beta agonist and 17-beta-oestrodial, form the journals i've read they are of 10nM each for all.

#10 bob1

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Posted 09 November 2009 - 05:09 PM

The levels of agonists and B-estradiol to use will vary according to the cell line. You will be best to titrate the appropriate level, checking for markers using PCR or protein responses.

The reason I say your time points are too short is that cells don't move very fast, you will not see much response by even 12 hours I suspect.

#11 jazmenia

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Posted 23 November 2009 - 07:39 AM

Hi,
I've done wound healing on MDA-MB-231 and MDA-MB-231-1833, and they have closure after 24hs.. some papers had closuer following 6hrs only :rolleyes:

No idea how they did it... i think it's just the wound measerment.. the bigger the scratch is, the more time they take to migrate... I guess MCF-7 are slower than the ones I used, coz their growth rate with me was slower compared to 231 and 1833..

anyway, adding EGF can make the healing faster... try it...

one question... how did you measer the motility distance????




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