The distribution of intracellular calcium chelator (fura-2) in a population of intact human red cells.
Using quantitative fluorescence microscopy of red cells loaded non-disruptively with 1-2.5 mmol/l cells of fura-2, we examined the distribution of the incorporated free chelator among and within individual cells. Cytoplasmic hemoglobin quenched the effective fluorescence yield of fura-2 by a factor of about 100. All red cells were found to fluoresce upon excitation at 380 nm, and the fluorescence intensities they emitted at 510 nm were approximately +/- 20% about the mean intensity, indicating a fairly uniform distribution of incorporated chelator among the cells. Red cells loaded with these high levels of fura-2 retained their biconcave shape, and a comparison between their transmission images at 415 nm and their fura-2 fluorescence images suggests that the concentration of fura-2 was also uniform throughout the cytosol. These results validate assumptions made in earlier experiments with non-fluorescent incorporated Ca2+ chelators, and demonstrate the feasibility of fura-2 and Ca2+ imaging of intact red cells, despite considerable quenching of probe fluorescence by hemoglobin.
Physiological Laboratory, Cambridge, UK.
V L
VL
Lew
Z
Z
Etzion
R M
RM
Bookchin
R
R
daCosta
H
H
Väänänen
M
M
Sassaroli
J
J
Eisinger
D002118:Calcium / Q000032:analysis; D048429:Cell Size; D004912:Erythrocytes / Q000737:chemistry*; D016257:Fura-2 / Q000032:analysis*; D006801:Humans; D008856:Microscopy, Fluorescence
D002118:Calcium; D016257:Fura-2