I want to learn to make my own. That's the fun in science for me is the problem solving and skill development.
Read that of course, but not up to the same quality I am getting from Mattek poly-D-lysine coated dishes. They use Corning or Falcon so there is something superior concerning the coating. Perhaps use 0.1M borate pH 8.5 during the coating process??? Wash dishes prior to coating??? I am looking for insight from someone performing these coating procedures and obtaining optimal results. I can throw 50 ug/mL on the dishes and wash, dry, etc except I am looking for some tricks to make adherence superior. Adherence, differentiation and viability are connected. With the Sigma protocol I am getting too much debris and a 2 day delay in differentiation as compared with the MatTek dishes. Also resistance to siRNA delivery and stress is superior in the MatTeks. I have tried a variety of plastic sources spanning Corning, Falcon, and several others. BD Falcon seems to be the best, but my coating is sub-optimal.
I read the Sigma protocol-- differs from the way we always did it. Work in sterile hood (UV going between steps is good) on a sterile cloth or liner. Have an entire sleeve all ready, remove one at a time, and as you coat the bottom, set the lid on top of it. I used to pull from the bottom so that lid remained in sleeve until it was place on top of the filled bottom.
You put in a ml (.5 for small 30-mm dishes), swirl to coat the surface thoroughly, place on lid; repeat etc. Do a bunch (I did a sleeve at a time)-- so that the first ones have probably been sitting wet for about a minute. One at a time, dump and flick dishes into a waste beaker, and place coated surface up near the laminar flow; sit until dry (3-5 min?). (You can also aspirate out, but keep tip at seam, avoid growth surface).
When dry, cover and you can store snugly in a sterile glass box (returning to sleeve not a good idea). Put in steril towel if they jiggle around, you don't want them coming apart.
Difference here: the Sigma protocol asks you to rinse; the drying is key. Lysine in either medium or BSS isn't going to leave a lot of residue, but it does overcome any electrostatic repulsion that a wet solution is encountering at the plastic surface.
Another way to do it: arrange all the dishes, take a 25 ml pipette and drop in a ml at a time, goes very fast. We also coated flasks; just ensure you've swirled over entire surface; takes much longer to dry.
What's the source of debris with Sigma so far? Strange...
Regarding culture dish type: not all plastic is the same, maybe your source has plasticizers or something you can leach out.
Hey Jason. I hope all is well. Did you get a good protocol? I'm in a similar state with THP-1s. I need them to stick a little better than they currently are and would like to test Poly lysine vs Fibronectin. Cheers.
Regarding the point about poly-D vs. poly-L-lysine: why use a form of amino acid not used or recognized by cells? Breakdown also means the cells can use the poly-L-lysine, and recognize it as a suitable adhesion surface.
Back in the pleistocene when ATCC ran cell culture courses at the Alton Jones Cell Science Center in Lake Placid, the belief was that the plastic surface needed to be treated as glass traditionally had been (called in vitro for a reason, folks!), using the treated surface to attract ions and biomolecules to keep cells happy and allowing them to flatten down and attach. Over time culture dishes were developed that were already coated or zapped; and with the push toward serum-free and more controlled conditions, the simpler coatings like poly-L-lysine had an advantage.
Would fibronectin be better? Most likely depends on the cell type, stage, and the fibronectin itself. We found in tadpole tail regeneration, the growth of new epithelial cells over the cut surface couldn't start until a layer of glycoproteins (not collagen) was excreted/laid down (https://www.ncbi.nlm.nih.gov/pubmed/?term=atkinson-k+dermoepidermal+regeneration). More to the present point, this paper is of high interest-- fibronectin mutations that promote differerent physiologic functions: Article Designed hydrophilic and charge mutations of the fibronectin...
Even a tiny change in your pH or buffer type might make your cells happier during attachment. Are you keeping them warm, not sitting in the hood for a long time? Perhaps doing streak tests of the different surface treatment options (dried in rows onto a single plate) and testing the cells for their preference is the best bet.
The way it's always been done isn't necessarily the best!
Michael Davis Jason Edward Hammonds We routinely use Poly-L-Lysine coated plates/dishes for our neural cultures with reproducible results every-time. We use 1mg/mL Concentration Poly-L-Lysine (dissolved in 0.1M Borate buffer -pH 8.4). Coating performed overnight, then wash 3x times with PBS and then equilibriate with your cell growth medium (5-10mins in 37-degress incubator) before plating the cells.
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