We seem to be having trouble getting 30-70 kDa poly-L-lysine (Sigma) to remain well bound to a glass coverslip. Cells (protists in this case) won't stick if the coverslip is rinsed multiple times with PBS, or even twice with 1% heat-inactivated serum in DMEM. In fact, the serum protein makes the poly-L-lysine "ball up" into ~2 micron spheroids on the surface, suggesting that it's not well bound.
We've tried a few things to solve this:
- from 1-10 mg/ml concentration poly-lysine in water
- 1 mg/ml dissolved in 50 mM pH 8.5 borate buffer
- drying the poly-lysine onto the coverslip before rinsing
- Corning versus Fisher sourced coverglass
In none of these circumstances do the cells remain well adhered, and the spheroids always form if the rinse contains protein. The odd thing is that one year ago (and one lab move) it worked acceptably well. Glass formulation has been suggested as a possible cause, though the Corning product numbers are the same before and since.
We would appreciate any thoughts, coating recipes, or other suggestions.
If you know of another non-specific adhesive surface for protists on glass, that too we'd love to know!
Hi William,
Not sure it is of any help (we use different cells), but from our own experience, striping/cleaning the glass coverslips with high concentration HCl and then washing thoroughly with sterile distilled water before to apply the coating helps.
I have coated with collagen with good results.
Dilute collagen to 50ug/ml in 0.02M acetic acid to the needed volume. Add diluted collagen, 5ug/cm² surface area, to dish or plate. Rinse dish 3 times with PBS or culture medium. ..
Best
I guess you have already checked the expiry date on the Poly L Lysine? I had the same problem last year (however another kind of Poly L Lysine (70-150 kDa) and primary cells) but when we bought a new lot of Poly L Lysine, it worked fine again. The one we use is only stable up to 2 years in -20 so if you haven't tried a new lot, it's worth a try.
Good luck!
Thanks for the replies so far! Nina: Our Poly-L-Lysine is new (though working with an older lot made no difference). Charles: We tried today coating with gelatin, but the cells didn't stick. These are picky little bugs!
Would love to hear some more ideas!
Hi William,
Not sure it is of any help (we use different cells), but from our own experience, striping/cleaning the glass coverslips with high concentration HCl and then washing thoroughly with sterile distilled water before to apply the coating helps.
We wash our coverslips with HNO3 50% and rinse 10 fold in H2O. Then rinse with non denatured EtOH 3 or 4 fold and dry overnight.
We coated 5 min with Poly-L lysine 0.01% final in H2O , Poly L lysine should be at room temperature when used. Remove the poly-L-lysine and let dry overnight at RT (or 1h at 50°C). Just before the experiment with the cells,we rinse the coverslips with PBS.
Good luck
Here is my protocol (for neuronal cell lines):
I use round glass coverslips 12-15mm diameter to grow cells and perform immunofluorescence at the end of culture. Coverslips are dipped in EtOH and dried then inserted into 24 or 12 well culture plates. Poly-L lysine (solution in H2O from Sigma, culture grade) is distributed to cover the bottom of the wells and incubated for 1 to 2 hours at room temperature, or at 4°C overnight. Then remove the polylysine and rinse the wells with PBS (without Calcium or Mg) 3 times (no serum added). At the time of use, remove the PBS and let the coverslips in the wells dry in a laminar flow cabinet and seed your cells on top.
Note 1: I do not further dilute the polylysine for the coating, but if it is removed in a sterile way, it can be recovered, stored at 4°C and reused several times.
Note 2: With cells that can digest poly-L-lysine, poly-D-lysine should be used as the attachment factor.
Note 3: Maybe use 70-150kDa polylysine (P4707 sigma) instead of 35-70kDa ?
Good luck
Hi William,
We had the same issue before and we fixed it when we acid-washed the coverslips.
Please check this protocol:
https://microscopy.duke.edu/sampleprep/washcoverslips.html
I hope it helps!
Alejandra,
We actually tried that already, and it didn't help.
Some clarification, though: When you say you "had the same issue," do you mean (a) failure of cells to stick, or (b) the formation of poly-L-lysine "balls" on the surface?
Replying to my own question, we eventually determined that poly-L-lysine precipitates with protein. Protein in the medium was thus stripping PLL from the coverslip (regardless of how it was repaired), appearing as spheroids on the surface. This was exacerbated by working at physiological temperature; at room temperature, the effect was reduced but not eliminated.
Laminin coating of the plate may be another option. Not sure if it will work for these cells.
Best!
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