Culturing cells on a slide - (Feb/07/2012 )

Dear Guys ,
In the beginning, I am very grateful for any one who will read my topic.

Secondly, I want to buy some culture wares for visualization of my cells under fluorescence or phase contrast microscope.
what I found due to reading some books and online:

*There are special type of culture slide such as
http://www.bdbioscie...y=4&mterms=true

*It is untreated.
I should treat the surface with Poly L lysine or collagenase as I am working with adherent cells
but I cant know the difference between these reagent and which on is the best.

* After culturing, Does the step of fixation is necessary, or adhesion to surface of my slide is enough.
* Shall I use the ordinary glass cover on my slide or use these precoated glass covers or precoat them by myself.
http://www.bdbioscie...rue&from=dTable

finally, does these steps differe if I will use a laser scanning confocal microscope?? Does any one has experience with it and ready to share??

At the end I am deeply indebted for those who will share their thoughts, experience with me.

Best Regards

Most adherent cells will grow on glass with no problem....no need to treat with poly L lysine. Some tricky cell lines (e.g. 293) might require poly L lysine so that they don't spontaneously detach. Chamber slides are nice to work with and let you use relatively less reagent. In the olden days, we used to just drop sterile cover slips into 6-well dishes and cells would adhere just fine to the untreated cover slips. When working with cover slips you need to make sure they are of the proper thickness for confocal microscopy (check with your local confocal expert). If you are going to be staining the cells with antibodies you will need to do a fixation/permeabilization step. My favorite protocol involves 3.7% formaldehyde (in PBS) for 10 minutes at 37C followed by 100% of -20C Methanol for 30 minutes. If you are looking at some fluorescent protein expressed by your cells, you can look at unfixed cells.

Doxorubicin is correct, cells will usually grow well on glass and the culture slides are a good option, though rather expensive. You do need to fix the cells if you are going to be doing an immunofluorescence protocol, in contrast to Doxorubicin, I use 2% parafomaldehyde in PBS for 10 min, wash lots to remove the PFA and then permeablise in 0.01% triton X-100.

I typically use 10-13 mm diameter coverslips in wells of a 24 well plate (they can't flip over in the wells, when you are getting them out), and square or rectangular coverslips work well in 6 well plates.

These procedures do not change for confocal.

Dear

bob1


doxorubicin,

I am very grateful for your precious comments.

You mean that I dont need to added any thing such as poly l lysine, or collagenase, or fibrocetin to ensure the attachment of my cells into glass slide (even culture glass slide are not treated, unless you buy it treated.

Secondly, so I can use sterile cover slip and drop it in my culture dish, then take it out, invert it, and put it in slide and watch my cells under microscope, is that what you meant doxirubicin,

I dont use ABs, so I guss fixation and permeabilization step is not required at least for now.

Thanks again

Based on what you have said, I can only assume that you want to observe a fluorescent protein that your cells express. If you are not going to be doing any kind of fixation step, then unfortunately, you can't just take live cells and smoosh them between two pieces of glass and assume that the cells will remain unchanged for the hours it will take you to snap pictures of your cells. If you really want to do live cell imaging, you should buy something similar to FluoroDish™ (World Precision Instruments, Inc.) so that your cells will remain in culture medium throughout the imaging steps. You will also have to regulate the temperature and possibly also the CO2 concentration.

I imagine you probably will just want to fix the cells to observe your protein, however, so that you can make "permanent" slides. To do this, you can sterilize coverslips by soaking them in 70% ethanol, then place them into 6-well dishes. Either let the ethanol dry or wash the wells one time with sterile PBS. Then trypsinize your cells and plate them in normal medium on top of the slides. After the cells are growing fine (24 hours), you can then wash away the medium, wash cells with PBS, and then fix/permeabilize the cells. You can probably get away with just adding -20C Methanol to the cells and letting them sit for at least 30 minutes. Next, remove the Methanol and wash with PBS. You can then pick up the coverslips with forceps and flip them over onto a normal slide. You will likely want to put a mounting medium (anti-fade reagent) in between the two pieces of glass and let the reagent dry at room temperature in the dark for a few hours. Then seal around the edges of your coverslips with clear nail polish, and voila, you're ready for confocal.

Not all cells stick to glass. Try staining them without any coating. But if you have trouble with them sticking, you need to coat them.