I would like to perform force spectroscopy on RBC using AFM. For this purpose I need 50ul of blood which would be diluted further in RPMI or PBS at pH7.4. In literature I see that disodium or dipotassium EDTA is used as anticoagulant. But we have only plane EDTA (EDS, Sigma) in powder form and 0.5M EDTA (Promega) in liquid form in our lab without sodium or potassium. Please let me know if they make any difference to the sample in terms of durability and morphology.
EDTA is used to stop coagulation by chelating the calcium. The metal ion EDTA interacts with is based on the acid form of the EDTA (Y-4 to Y-1) so to chelate Ca you have to be in the right pH range, the counter ion only really matters for electrochemical studies where ion concentrations becomes important.
Here is a description of how the chaltion happens for determining water hardness, a fairly close example for what you will work on.
http://chemistry.bd.psu.edu/jircitano/Water05.pdf
I think EDTA is just too hard to dissolve. And how will they use PBS, with EDTA disodium will increase the amount of Na and could change the pH . So they should use dipotassium EDTA or tripotassium because are the best for the cell morphology.
Hi Roberto, it is difficult to dissolve EDTA powder at pH 7.4 but we also use 0.5M EDTA liquid at pH 8 which easily dissolves but changes the pH of solution. So I adjust the pH of final solution to 7.4 which will be used for dilution of blood. In the final solution 6mM or 1.8mg/ml EDTA is present.
1. Would this solution let the cells be in their native state?
2. What would be the average amount of albumin in blood as this seems to be also responsible to help the RBC remain in biconcave shape?
3. Is the quality of blood collected by finger prick method similar to the blood taken from veins?
4. Would it be harmful to perform finger prick collection of blood on same person on a daily basis?
This information would be very valuable for me.
EDTA (acid) is poorly soluble in water. At 20 deg C, staurated solution of EDTA will have 0.5 g/l concentration. Molecular weight is 292.2. So forget to have EDTA solution of 0.5 moles/L ! Its solution is acidic; pKa is 1.782, far away from blood pH. Only by adding NaOH with titration at pH of about 8.0, you can make 0.5 M EDTA. Either di-Sodium or di-Potassium EDTA solution will have pH in the range of 4 to 5. So, at pH 8, it will be almost tri-Sodium or Potassium EDTA solution.
It is not necessary to use EDTA in such experiments. Just coat the pipette tip with heparin solution before blood collecting, it will be enough.
Good luck!
I agree with Denis, as heparin is a physiological anticoagulant and negligible quantity is needed.
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