scrape loading dye transfer

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vserra04b
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scrape loading dye transfer

I am using lucifer yellow and my cells have Cx43 junction. the 1st time I tried it- the cells transferred the dye. Now I am having no luck in getting the cells to transfer the dye. I have used sterile PBS, HBSS as well the cells conditioned media to rinse and dilute the LY in and am getting no luck.....any ideas as to why or how this could happen?

neil
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Hi there, i've been doing a

Hi there, i've been doing a bit of scrape loading to quantify gap junction communication. What cells are you using?

I can't see why PBS isn't working, it should be fine. What is the calcium concentration of your PBS as Ca2+ will close all gap junctions? What concentration of Lucifer yellow do you use?

We use 4% LY in PBS. Layer over the cells, scrape, incubate for 5 minutes, remove and wash extensively with PBS plus 1.5mM Ca2+ to stop dye spread. Record pictures within minutes. This protocol hasn't failed us.

Have you checked recently that the cells do still express Cx43?

vserra04b
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Hello,

Hello,
Thank you for responding.

I am using a relatively new cell line- normal human thyroid, HTori-3.
I am using Dulbeccos PBS with a CaCl2 concentration of 0.1 g/L

I have used varying concetrations of LY. The best result I have had is when I use it straight even if technical support says to use it 1:10. The concetration of LY is 100mM from Invitrogen. I have used 1:250, 1:10, 1:3 and straight concentration. My timepoints varied between 1min to 10min.

I rinse the cells in DPBS 1 time, make a scratch with a pipette tip down the middle of my 35mm Petri dish and release the dye at the same time. Then rinse 2 times with DPBS and keep the cells in the 2nd wash to take pics.

The first time I performed this assay- I used PBS with azide, and a concetration of 1:250. I think that the transfer might have been due to cytoplasmic bridges. These cells are supposed to have Cx43 junctions so it should work.
Since the dye is not spreading I am not stopping the dye with Ca2+

We received these cells in RPMI media with 10% FCS and we changed the media to DMEM:F12 (50:50 v/v) with 5% bovine calf serum & 6 hormonal mix. We did this to 'normalize' the cells.

I will try your protocol.