Dear Chia-Wei Phan, I don't want to create more confusion, though each suggested coating either with collagen I or IV or with laminin should work well for attachment of your cells. However, I fully agree with the comments of Justin, David, Abbas, and Daniel in one way or the other that (1) the mentioned ECM molecules may have different influences on your cells concerning ahhesion complexes, migration, growth, and direction of differentiation and (2) indeed laminin should be more favorable on the developmental and physiological aspect. Just a few more remarks to laminin, this protein family consists (like collagen) of many different isoforms. So, I assume the source of 'your' mouse laminin is the EHS tumor (also source of the complex 'matrigel'). This should be laminin 1 or better laminin111 (new actual nomenclature defining the alpha, beta, gamma chains) appearing first in development and persisting in some internal organs/ sites through adult life. In addition, like many other laminins this type is tightly associated with the crosslinker or adapter molecule nidogen (1 or 2) promoting basement membrane formation. Nidogen can also enhance signaling effects of laminin on cells, as shown for breast epithelial cell differentiation by Mina Bissell and Rupert Timpl years back. There are several recent reviews on the subject. Good luck.

Hi!

I've been working with neuroblastoma cells for 5 years and I don't use any coating. This type of cells are very adherent.

Maybe us better collagen.

Dear Carla, Thanks. For the cells it adheres perfectly in flasks/ well without coating. I am currently using the cells for a specific assay using a kit from a manufacturer. At first no coating was used but the results were poor and non-reproducible. The manufacturer gave a feedback that extracellular coating matrix (ECM) protein is crucial to coat the well (regardless of adherent or non-adherent cells). So, in this case, collagen will be good as suggested by you? I am eyeing for BD Biosience collagen. Or any brand is fine?

I'm not really competent in this matter. You'd better contact with these issues to Vladimir Pichugin or Roman Surmenev.

I agree with Danilo

Ok guys, thanks! will share it here once I have tried it out

I would also use collagen I. I used both BD bioscience and Sigma collagen I. They were equally good for my purpose.

I will not be able to say which coating agent will be the best for your application. However, this question illustrate some other aspects which can affect the study of cell metabolism, and which might be considered for the right choice of such a coating .

Cell membrane present topographic distribution of adherence linked to local molecular structure and composition and to specific local density of van der Waals binding energy. and is always depending on the material on each side of an adhesion interface. Further more adhesion can be achieved with a high number of low energy adhesion bridges, or with a reduced number of adhesion bridges of higher energy.

In a first step can be looked at the topography of the cell membrane surface in order to get some idea which sort of coating material is compatible with the considered cell membrane. High resolution scanning microscope can be used for this purpose.

However, local high van der Waals adhesion energies can induce some transverse transient polarization which can affect the molecular structure of the cell membrane and of the cell material inside. Therefore, in addition to the selection of an adherent coating agent, should be looked at the morphology of the surface with which the local adhesion energy is provided, and here preference should be given to the case where the adhesion is obtained with a distributed higher number of adhesion points which have each of them lower van der Waals or lower chemisorbtion energy.

This sort of effects explains the existence of Monod centers which react to some specific molecular compounds, explains the olfaction effect, and why high sensitive olfaction function can specifically detect some cancer cells and some of corresponding molecular residues.

You can get more about these effect in published papers SENSOR AND ACTUATORS B Vol 121 (2007)' 436-444

Type IV collagen from human placenta (Sigma-Aldrich C5533) can be used in your study. It has been shown that a number of different cell lines show strong adhesion on collagen surfaces.

For your study, I think rat tail type I collagen is better choice than the coated one.

I would suggest laminin as it is more plentiful in the CNS than collagen, therefore making laminin more physiologically relevant. Though I know that other neuroblastoma cell lines do grow well on collagen (e.g PC12 and NS1 cell lines).

agree with Danilo and Punitha - Rat tail type 1 collagen -

We're using either laminin or fibronectin successfully as coating agents for human neuroblastoma cells

The better extracellular matrix membrane will be collagen material based membrane or other biocompatible material based membrane. I developed pure collagen nanofiber membrane, some research group developed collagen coated nanofiber membrane, other group developed collagen casted membrane, all of these membrane could be your choice.

I am using laminin to attach human glioblastoma neurospheres onto coverslips and fibronectin for human adherent cancer cells that do not attach well on glass coverslips.

It is dependent of your stem cell source

Membrane coating proteins of the Extracellular matrix are different as the ECM itself (skin, bone, teeth, the blood or the basal lamina?). Therefore, what you should better use depends on (1) the subject and (2) the specific aims and objectives of your research. This is because different glycoproteins have different; and specific receptors to different collagen types. This is a condition to not only their major role in chemical signalling between cells where they bind various secreted signal molecules such as certain protein growth factors, but also in their other function such as simple mechanical support functions, modulation of diffusion, and stability of various cytokines, effects on cell adhesion, motility, and proliferation; influence on differentiation and tissue morphogenesis. So, over to you!!!

I think you must choose the one is more physiologically relevant for your study. I use Laminin because I work with muscle satellite cells… os it is relevant for me. As you can see from all the reply every person has a personal view on the issue. I recommend you to do some reading about the composition of the external matrix in where you cells (or the most closest type of cell to the ones you are going to use) normally growth and use the one most close to the physiological conditions. However, I have to admit that collagen is a quite general good option in where most cell lines are happy to stick and growth. However, be careful, the cellular matrix can affect your results in ways you don't know. So, the closest your experiment in to physiological conditions the better to interpret your results (and that includes the matrix in the cell culture).

Dear Chia-Wei Phan, I don't want to create more confusion, though each suggested coating either with collagen I or IV or with laminin should work well for attachment of your cells. However, I fully agree with the comments of Justin, David, Abbas, and Daniel in one way or the other that (1) the mentioned ECM molecules may have different influences on your cells concerning ahhesion complexes, migration, growth, and direction of differentiation and (2) indeed laminin should be more favorable on the developmental and physiological aspect. Just a few more remarks to laminin, this protein family consists (like collagen) of many different isoforms. So, I assume the source of 'your' mouse laminin is the EHS tumor (also source of the complex 'matrigel'). This should be laminin 1 or better laminin111 (new actual nomenclature defining the alpha, beta, gamma chains) appearing first in development and persisting in some internal organs/ sites through adult life. In addition, like many other laminins this type is tightly associated with the crosslinker or adapter molecule nidogen (1 or 2) promoting basement membrane formation. Nidogen can also enhance signaling effects of laminin on cells, as shown for breast epithelial cell differentiation by Mina Bissell and Rupert Timpl years back. There are several recent reviews on the subject. Good luck.

Dear Chia-Wei Phan

Laminin induces neural differentiation and neurite outgrowth via α6β1 integrin receptors so laminin in your case is better candidate. However, we showed that type of DI water in media is so important in cell attachment on flask. If you make your media please JUST add DI water from a company (milipore) and not from your laboratory. It is so important owing to effect of pH and some residue in water.

I agree that Laminin is most likely the best. One thing to keep in mind is that protein tends to bind with variable efficiency to glass. This means that the affective concentration may be much lower than your solution concentration. There are multiple treatment that can improve coating (Bind sylane for example works well). Another thing to consider is the rigidity (stiffness) of the substrate. The normal environment is soft while glass is very rigid. For fibronectin, I find that first coating the glass with gelatin (denatured collagen. 1mg/ml) and then allowing the FN to bind naturally to this first coat produces very different cell behavior even when cells have no receptor for collagen.

Collagen type I is the most commonly used coating protein as it keeps the cells in their original morphology . While the cells grown on plastic usually attain the flattened morphology and stick to the bottom of the tissue culture dish. In my opinion neuroblastoma cells should grow fine on the plastic culture dishes but if you want to be sure that your culture conditions are same as invivo you can try collagen type I as I have used that for growing primary chondrocyte culture.