I am performing AFM force spectroscopy on red blood cells by immobilizing them on glass surface. I clean the glass coverslips by sonication in soap solution, Milli Q, ethanol, acetone, ethanol for 10min each. Then I dry them and add 0.01% Poly-L-Lysine 150000-300000Da for 30min and wash them with MilliQ and dry under nitrogen. Then add 20ul RBC which is resuspended in RPMI1640 medium. Then wash the surface with RPMI and add fresh RPMI on the glass coverslip which is in AFM liquid cell.the cells crenate in 15min. For a typical experiment I would like them to be in good condition for atleast 2hrs. Please let me know how to solve this issunnn
Hello Sumanth.
I do not know the final volume of the cell suspension, is it 20ul drop of RBC? Repeated drying and additions of medium would accumulate high salt concentration, leading to becoming more hypertonic solution. Use a larger volume of dish (i.e., 30mm dish containing a coverslip plated with RBCs).
Rapid evaporation and frequent additions of medium should be avoided by using many prepared RBC-coverslips cells in CO2 incubator by changing the cell-covered slips.
Use a simple isotonic salt solution, such as 4mg BSA/ml HBSS.
Increase volume greatly the cell chamber volume. You are coating the coverslip + RBCs with salts, amino acids, other organic (FBS?) time after time whenever you add more medium.
Decrease the ambient temperature to delay evaporation.
Regards.
High ion strength induces crenation. Low pH can give the same effect. As Dr. Ho Lee said, it can be just an effect of drying - decreasing of volume with increasing of ion concentration.
We have used poly-l-lysine slides from Thermo and just conventional PBS (pH 7.2 - 7.3) for the same AFM experiments. All was Ok.
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Good luck!
Crenation in 15 minutes seems a typical osmotic problem. Try to put them in different salt (NaCl) concentrations below and above physiological and see where is the stable point. For us, on PEI coated glass coverslips they usually can be kept and measured for 2-3 hours, however sometimes they blow up with similar time coarse like crenation in your case.
Luck!
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