how to use poly L-lysine for adherence of neutrophils?
Treat the coverslip with diluted poly L- [with H2O to 0.01% (v/v)] at RT for 30min, then wash coverslip with H2o and air dry it. After that the coverslip is ready for use.
Generally, you can modify a glass substrate for cell attachment by incubating the substrate with 1mg/mL of poly-L-lysine for an hour or even over night.
Below is Sigma-Aldrich's protocol for Poly-L-Lysine mediated Cell Attachment:
http://www.sigmaaldrich.com/technical-documents/articles/biofiles/poly-lysine-product.html
However, poly-L-lysine is not a good substrate-modifier for cell migration because most likely the cells will be stuck and unable to move about on the poly-L-lysine. For neutrophil (human or mice) migration works, fibronectin and ICAM-1 are better candidates of substrate-modifier (more physiologically-relevant). BSA also works if you are looking for some cheap protein to use (BSA has been reported to be a ligand for Mac-1 integrin. Reference: Anderson, Donald C., et al. "Contributions of the Mac-1 glycoprotein family to adherence-dependent granulocyte functions: structure-function assessments employing subunit-specific monoclonal antibodies." The Journal of Immunology 137.1 (1986): 15-27).
Actually, neutrophils will also non-specifically adhere to glass slides or coverslips even without poly-L-lysine, because of the negative charge of glass.
Note: If there are proteins (especially serum) in your media (for hosting the cells), the proteins will adsorb onto the glass slides or coverslips to reduce cell adhesion, but that generally takes some times.
PS: I have not used poly-L-lysine before for my work, but from past experience I know that neutrophils (mice and human) are generally sticky, and will stick to fibronectin, BSA, ICAM-1, and bare glass.
Thank you very much Henry, its really detailed and complete answer.
Neutrophils crawl onto glass enthusiastically provided that the glass is properly cleaned . Slides and cover slips need to be treated with dilute HCl to remove alkali and then thoroughly washed in water and dried.
The best way of then making the monolayers is described in detail in my first ever paper - Immunology 4:142-152 (1961). In brief, blood is taken into edta and 0.6% dextran. The red cells are allowed to settle out at 37 degrees in an inclined rack and the platelet and white cell rich plasma removed. two drops of this are recalcified and put on the clean slide and incubated in a moist chamber for an hour at 30 degrees. The plasma clot is then removed and the white cell monolayer kept moist with a drop of serum.
Article A two-stage indirect L.E. cell test
Thank you very much Lachmann sir. Eminent scientist like your caliber sparing some time to guide us really motivates a budding scientists. I want to study NET formation in response to Lesihmania donovani. If I find any difficulty I will look forward for suggestion from you sir. Thank you once again.
I recommend that you try Shi-fix coverslips from Shikhar Biotech
http://www.shikharbiotech.com/products/shi-fix-coverslips/
.They work on a large variety of cells. You can immobilize the cells by just allowing them to settle on the coverslips for 30 mins.
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