I am having some troubles with PBS solution I am currently using to work with erythrocytes. When comparing the erythrocytes suspensions in PBS (pH = 7.4; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4) and in water (complete hemolysis), the PBS shows 20% hemolysis when, ideally, it should be close to 0%.
Briefly, the degree of hemolysis is determined by measuring the absorbance of the suspensions at 550 nm.
How can I assess the osmotic stability of an erythrocytes suspension?
Thanks!
Dear Dr. G. Martins,
To know how to assess and maintain the osmotic stability of an erythrocytes suspension, please read an useful paper of P.C. Chikezie, entitled “Comparative in vitro Osmotic Stability of Three Human Erythrocyte Genotypes in the Presence of Quinine and Chloroquine Phosphate”, published in Asian Journal of Biochemistry, 6: 55-64 (2011). The author presented in vitro study to ascertain the tendency of two quinoline (quinine and chloroquine phosphate) drugs to interfere with osmotic stability of three human erythrocyte genotypes, namely, HbAA, HbAS and HbSS. Spectrophotometric method (like yours, Dr. Martins) was used for determination of the capacity of the erythrocyte genotypes to withstand osmotic stress in the presence of separate increasing concentrations (0.2, 0.4, 0.6 and 0.8 mg%) of the two quinolines. Major point related to your request is that Dr. Chikezie utilized the Mean Corpuscular Fragility (MCF) index for all erythrocytes he used.
Best wishes,
Dr. I. Tsyrlov
Dear Goncalo
Did I understand you correctly?
You are measuring "the absorbance of the SUSPENSIONS at 550 nm."? You need to test supernatant after centrifugation of the suspension.
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