We are working with single cell layers on microscope slides. The cells need a surface coating to adhere to the glass, which is currently a polylysine 140 kD variant. We still have attachment problems. Does anyone have a suggestion to improve the cell adhesion to the glass? We use clean, new microscope slides which have been cleaned extensively. Appreciate your suggestions.
It will depend on your cell type. However as a general rule, if the biological methods aren't working and you have access to a physics or engineering department with a plasma reactor, a short oxygen or air plasma should improve adhesion by 1) making the surface more hydrophillic and 2) introducing a small amount of topography to increase adhesion. Otherwise, gelatin coating, fibronectin or laminin coating, or even collagen coating should work. However if your glass is too hydrophobic or too hydrophillic you will struggle to get any biomolecule to adsorb.
Finally check you're using the correct seeding density, for some cell types if there aren't enough other cells around then they might initially attach but they will very quickly die off. Unfortunately without knowing the specific cell type it's difficult to give much more advice.
Depending of the type of cells, if you add CaCl2 (final concentration around 3mM) and MgCl2 (at about 11mM) in the cell solution, that will help the cell layer to adhere to the slide. I used to clean also the slide prior coating with HCl 10%. Good luck and tell us it that work!
Dear Maryline Bossus,
Thanks for your suggestion, but the cells we are using do not permit the use of high CaCl2 concentrations. Otherwise, it would have been a good and useful suggestion.
Thanks!
have you tried to coat your slides with 100% FBS for at least 30 mins before use??! this may help some how.
With H9C2 cells we have 10% FBS in the media (DMEM) to facilitate adhesion. With primary culture of adult cardiomyocytes I have seen the use of gelatin coating of glass coverslips to facilitate adhesion.
Good luck
What is your cell type? Some cell lines (especially neuronal cell lines) need to prior coating of surfaces with poly-L-lysine, collagen etc...
hey you can coat your slides with 0.2% gelatin in PBS for 20 mins, air dry it. After that add your cells in the presence of the FBS ( neat) on the cover slip ony for 3-4h to allow the cells to attach followed by the addition of the extra media to make the final concentration of the FBS 10% or as required. Incubate for overnight further n I am sure your cells will attach properly.
It will depend on your cell type. However as a general rule, if the biological methods aren't working and you have access to a physics or engineering department with a plasma reactor, a short oxygen or air plasma should improve adhesion by 1) making the surface more hydrophillic and 2) introducing a small amount of topography to increase adhesion. Otherwise, gelatin coating, fibronectin or laminin coating, or even collagen coating should work. However if your glass is too hydrophobic or too hydrophillic you will struggle to get any biomolecule to adsorb.
Finally check you're using the correct seeding density, for some cell types if there aren't enough other cells around then they might initially attach but they will very quickly die off. Unfortunately without knowing the specific cell type it's difficult to give much more advice.
I would suggest first to make sure that your slides are properly treated : The best way I know is to wash them in 1M HCl overnight at 60C on a magnetic stirrer, then 6h wash vigorously with DDW again on magnetic stirrer followed by another 6h wash with 70% Ethanol and finally drying inside a laminar flow hood. (from now on your slides are clean and sterile and ready for coating).
Once the slides are dry you can coat them with Gelatin (1%), Fibronectin (100 ug/ml) (This is my choice), Collagen I (30 ug/ml), FBS which all work perfectly. If you can not use any of them just pre-wet the surface of the slide with your 10% FBS containing medium and leave it to somehow dry inside the laminar flow hood.
Knowing the cell type really helps. In general, it's rare for cells not to stick to fibronectin coating. If they are not sticking to an ECM coating, then the coating itself was no good.
One word: fibronectin. 5-20 ug/ml in PBS++ for 1 hour at 37C, 4 hours at room temp, or overnight at 4C.
If your cells are expressing LFA-1 or Mac-1 I would try coating the glass with ICAM-1
Thank you all for your contibutions. I will test some of them in the near future (weeks). Particularly intriguiging was the contribution by Dr. Smith. Could you elaborate on the use of the plasma for making the glass surface more hydrophilic? I do have access to such a facility, so if worthwile, it could speed things up.
Thanks so far to you all!
Depending on which plasma, and operating parameters, you use the plasma will etch away the surface introducing a slight amount of topography which will affect the contact angle. Also because of the fact that your coverslips are in an charged environment you can get addition of functional groups to the surface. For example tissue culture plastic is plain polystyrene that is injection molded into multiwell plates. These are then exposed to a plasma (air, oxygen, nitrogen, mix of the above depending on the manufacturer) which introduces oxygen and nitrogen into the plastic. The energy of the molecules bombarding the surface also breaks the bonds in the polystyrene which introduces other functional groups.
I have a question for everyone in this forum. I am differentiating megakaryocytes from mouse bone marrow aspirates and then checking for proplatelet generation from these differentiated megakaryocytes. I want culture these megakaryocytes in fibronectin coated coverslips and then carry out immunofluorescence. These cells a non-adherent and float around in culture. What concentration of fibronectin should I use? We have some human fibronectin solution in the lab. Do mouse cells attach to human fibronectin?
Kasturi, start with 10-50ug/mL coating FN. FN preps vary greatly; try both extremes as an initial test to see which will work. Mouse cells will adhere to human FN.
Dear Louise Elizabeth Smith,
I am working with bare silicon sensor chips. I have been advised to make sure no large static charges are accumulated during the plasma treatment. Since I do not have access to a plasma treatment set-up, I have been thinking to make my own. This by putting my silicon chips in a small Faraday cage, put this cage in a small, plastic excicator, and then treat this excicator in a kitchen type microwave oven. After evacuation to below 1 mTorr, the microwave will be activated, resulting in a mostly oxygen based plasma. My first try will be 30 seconds at full power and see what this yields. At the moment busy with modifying the kitchen microwave (A Philips 1000W type).
Kind regards, Harrie Verhoeven
For non adherent cells, there is also the option of centrifugation.
To do this you need special centrifuge accessories to carry the slides (baskets) . One commercial name of such equipment is called cytospin.
There are also accessories that can carry well plates.
The centrifuge rotor it is not necessary to be exchanged, you can buy only the baskets.
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