Determination of the lowest concentrations of aldehyde fixatives for completely fixing various cellular structures by real-time imaging and quantification

Abstract

The effectiveness of fixatives for fixing biological specimens has long been widely investigated. However, the lowest concentrations of fixatives needed to completely fix whole cells or various cellular structures remain unclear. Using real-time imaging and quantification, we determined the lowest concentrations of glutaraldehyde (0.001-0.005, ~0.005, 0.01-005, 0.01-005, and 0.01-0.1 %) and formaldehyde/paraformaldehyde (0.01-0.05, ~0.05, 0.5-1, 1-1.5, and 0.5-1 %) required to completely fix focal adhesions, cell-surface particles, stress fibers, the cell cortex, and the inner structures of human umbilical vein endothelial cells within 20 min. With prolonged fixation times (>20 min), the concentration of fixative required to completely fix these structures will shift to even lower values. These data may help us understand and optimize fixation protocols and understand the potential effects of the small quantities of endogenously generated aldehydes in human cells. We also determined the lowest concentration of glutaraldehyde (0.5 %) and formaldehyde/paraformaldehyde (2 %) required to induce cell blebbing. We found that the average number and size of the fixation-induced blebs per cell were dependent on both fixative concentration and cell spread area, but were independent of temperature. These data provide important information for understanding cell blebbing, and may help optimize the vesiculation-based technique used to isolate plasma membrane by suggesting ways of controlling the number or size of fixation-induced cell blebs.

Fixation‐induced cell blebbing on spread cells inversely correlates with phosphatidylinositol 4,5‐bisphosphate level in the plasma membrane

“…2H). However, compared with 4% paraformaldehyde, 3% paraformaldehyde induced much smaller blebs which is consistent with our previous report [17].…”

“…On the other hand, at low temperature (e.g., 4°C) the behavior of enzymes, protein-protein interactions, and intracellular signaling will generally slow down, including the cortex-related behaviors. However, aldehyde fixatives at low temperature still can induce bleb formation dramatically in number and size [17]. These observations suggest that the actin cortex has potentially been fixed immediately after the addition of the fixative solution particularly at relatively high concentrations and therefore the local rupture of the cortex was not, or at least not majorly, responsible for bleb formation although there were no strong evidence to exclude the pos-sibility that aldehyde fixation can cause the local rupture of the cortex.…”

“…Then, a ratio of the total cross‐sectional area of all blebs to the spread area of the cell and the average area ratio per cell were calculated. Previously, we found that the sizes of blebs on a cell inversely correlate with the spread area of the cell [17]. Therefore, analyzing the ratio of the total cross‐sectional area of all blebs to the spread area of the cell is able to as much as possible exclude the effect of the degree of cell spreading.…”

AFM visualization of cortical filaments/network under cell-bound membrane vesicles

“…It is well established that the cytoskeleton closely linked with the inner leaflet of the plasma membrane can provide the plasma membrane with the mechanical strength to withstand the stress or shear forces from the outside environment [22,23]. If there was a cytoskeletal network associated with the cytoplasmic surface of the vesicle membrane and the cytoskeleton became rigid due to the aldehydeinduced fixation [24], the cell-bound membrane vesicles should not be so easily depressed by AFM probe or broken by air drying. Third, we indeed observed filamentous structures or network inside individual vesicle-derived pits (i.e.…”

Effects of membrane lipid composition and antibacterial drugs on the rigidity of Escherichia coli: Different contributions of various bacterial substructures

“…UV-5100 spectrophotometer (Metash Instruments, Shanghai, China) was used to measure the OD value at 600 nm. The cell viability was determined by the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)) assay (Shao et al, 2013;Zeng et al, 2013). Briefly, after the bacteria were treated with the antimicrobial drugs and rinsed twice with PBS, 100 ml of the treated bacteria and 20 ml 5 mg/mL MTT (Amresco, Solon, OH) were added into each well of a 96-well-plate and incubated for 4 hr.…”