Stability of Cells Fixed With Glutaraldehyde and Acrolein

Abstract

Dielectric techniques have been shown to be useful in studies of the effects of osmium-fixing procedures on the stability of erythrocytes (Carstensen and Smearing, 1967 ;Carstensen et al ., 1969) . In this report a similar approach has been used to investigate calf erythrocytes fixed with glutaraldehyde and acrolein . Both agents can produce stable cells and preserve as an insulating barrier all but a fraction of a per cent of the membrane . METHODS AND MATERIALSCalf blood collected with sodium citrate as an anticoagulant was washed twice in 0 .145 M saline to remove plasma and the huffy coat . Glutaraldehyde FixationTwo forms' of glutaraldehyde were used in these studies, the first, a highly purified monomeric form, and the second, a condensed form :(a) TAAB2 glutaraldehyde was purified by shaking with charcoal and distillation (Fahimi and Drochmans, 1965) . As received, this material always has considerable spectrophotometric absorption at 235 nm, which was completely removed by this purification procedure . Immediately before use, the stock solution (24 .4%) was filtered through a Diaflo UM-05 membrane (Amicon Corp ., Lexington, Mass.) at 30 t In addition, a single check was made using a technical grade (Baker) glutaraldehyde . Immediately after fixing, cells from this preparation had a low frequency effective dielectric constant of between 200 and 300 . Thus, even though the cells were superficially stable and appeared normal in the light microscope, from a dielectric point of view their membranes had almost vanished . 2 TAAB Laboratories, Emmer Green, Reading, England . psi and then diluted to obtain the final fixing solution containing 2% glutaraldehyde, in 0 .14 M NaCl, 0 .005 M sodium acetate, and 0.005 M CaC12 (pH 7) .(b) Polysciences3 EM grade glutaraldehyde as supplied (8% aqueous solution, pH 7) had a large peak in the absorption spectrum at 235 nm . From ultracentrifugation it appears that more than 90% of this glutaraldehyde is condensed to dimer or higher species . A 2% fixing solution was made by diluting this reagent directly with isotonic (1/15 M) phosphate buffer (pH 7) .For fixing, one part of packed cells (hematocrit -0 .80) was mixed with 10 parts of fixing solution and held for 10 min . The cells were then centrifuged and washed two times in 0 .145 M NaCl to remove excess glutaraldehyde . Acrolein FixationAcrolein was purchased from the Shell Oil Co., New York . As supplied, the raw acrolein contained approximately 0 .1 % hydroquinone as a stabilizer, and was partially polymerized . Before use in these experiments, the acrolein was distilled at 55°C at ambient pressure in a rotary evaporator using a constant flow of dried nitrogen or helium gas . The distillate was collected in a flask, cooled by an acetone-dry ice mixture (Aldridge and Wickens, 1971) . Two acrolein preparations were used in these studies ; one was a 2% solution without stabilizer, and the other was a 10% solution with stabilizer : (a) The unstabilized acrolein in these preparations was stored (no longer than 3 weeks) at -...

Contraction of gut smooth muscle cells assessed by fluorescence imaging

“…The present method may thus provide more accurate information with regard to the contractility response to various agents. However, because the fixation by acrolein was reported to result in 5e10 % reduction in cell volume (15), it should be noted that the present method might underestimate the contractility compared to the evaluation method using with real-time microscopic observation of living cells.…”

Saturation transfer in human red blood cells with normal and unstable hemoglobin

“…Consistent with these findings, it was also reported that glutaraldehyde fixation of RBCs results in 5$10% reduction in RBC volume. 48,49 Action spectra, CR spectra and CR ratio vs f 2 (ppm) Ð Correlation with physical states of protein± water and protein±protein interactions RBCs (Hb Yokohama). CR spectra for dense cells of RBCs (Hb Yokohama) and normal RBCs were similar to those for protein gels and BSA solution, 11,24 respectively, after correction for protein concentration effects on CR values, indicating strong association or aggregation of unstable Hb molecules in RBCs (Hb Yokohama).…”

Effects of glutaraldehyde fixative osmolarities on smooth muscle cell volume, and osmotic reactivity of the cells after fixation

“…Elbers, 1966;Fahimi & Drochmans, 1965;Korneliussen, 1972;Bone & Ryan, 1972;Brunk & Ericsson, 1972;Thureson-Klein et al, 1975;Tisdale & Nakajima, 1976;Boyde et al, 1977). This is in contrast to some reports (Morel et al, 1971;Carstensen et al, 1971 ;Penttila et al, 1974), which stated that cells become osmotically inactive after GA fixation. Our tissue strip study showed that SMC were behaving like an osmometer after GA fixation, in that increasing the osmolarity of the superfusate (through the addition of sucrose to the buffer) increased the tension (shrinkage), whereas tension decreased when the osmolarity of the superfusate was lowered.…”