The simultaneous use of Hanks' and Hepes buffers in the preparation of human leucocytes for SEM observation

Abstract

Leucocytes were isolated from the peripheral blood of normal individuals and prepared for SEM observation. The addition of the buffer Hanks and Hepes to the glutaraldehyde fixative was found to maintain the initial ambient pH throughout cell manipulation, and to allow satisfactory preservation of surface architecture.

The use of Hanks'‐pipes buffers in the preparation of human, normal leucocytes for TEM observation

“…In some cases vacuoles occupied most of the cytoplasmic space and contained amorphous material and membranous structures in a relatively electron-transparent medium. Much data originating from SEM studies have been interestingly controversial and advantageously contributive because of modifications of human leucocyte surface morphology caused by the variations in the techniques used to prepare these cells for electron microscopy observation (Abugaber et al, 1981;Barber & Burkholder, 1975;Corvellec et al, 1983;Lalague et al, 1982;Polliack, 1977Polliack, , 1978Polliack, , 1979Renau-Piqueras et al, 1980;Roath et al, 1978), yet very few reports on the effects of preparatory conditions on the ultrastructure of peripheral blood leucocytes have been available. We have investigated the use of (i) 4% glutaraldehyde (G)+ RpMI-1640 medium, (ii) 4% G+RPMI-1640+0.05 mol sucrose, (iii) 4% G+Hanks, (iv) L4 % G + Hanks + 0.04 ,moll sucrose, (v)] 4%dG + PBS] (phosphate ,buffered saline), (vi) light- Fig* 3.…”

“…The material obtained was adjusted to 3.0 x 106 cells/ml with Roswell Park Memorial Institute (RPM1)-1640 medium and 5 ml of this suspension were added to the following culture medium : (2 x ) RPMI-1640, 25 ml + autologous serum, 5 ml + penicillin (1000 IUjml), 5 ml + streptomycin (1000 IU/ml), 5 ml + phytohaemagglutinin, 0.5 ml + pokeweed, 0.5 ml. This preparation was placed into a 250 ml DeLong culture flask containing polycarbonate membranes (previously coated with carbon, covered with a thin film of poly-Llysine, 1 mg/ml, and RPMI-wetted) and incubated for 48 h at 310 K. In a 100% humidity chamber the membranes were then washed twice with deionized, Millipore-filtered water (DMFW) and twice with DMFW containing HaPiS buffers at 296 K, pH 7.4 (HaPiS buffers: Hanks (Ha, see details in Lalague et al, 1982)+pipes (Pi), 2 5 x molt-sucrose (S), 4.5 x 10-2 mol; the HaPiS buffer formulation was devised in this laboratory).…”

“…Meticulous studies of the preparatory conditions of human peripheral blood leucocytes are of utmost importance for proper scanning electron microscopy (SEM) investigation of cell surface architecture or transmission electron microscopy (TEM) observation of ultrastructural cellular characteristics. We have recently reported on what we believe represent quite advantageous chemical combinations in the treatment of human peripheral blood leucocytes for SEM studies (Lalague et al, 1982;Corvellec et al, 1983); the buffer-fixatives used for the SEM preparations were found to be not only quite efficient for these experiments, but also suggested their possible use in the preparation of cultured, normal human leucocytes for TEM examination. The purpose of this brief communication is to report on the use of Hanks'-pipes buffers, and their advantage, in the general treatment of leucocytes for T E M examination.…”

Tannic acid‐ and thiocarbohydrazide‐mediated osmium tetroxide binding in the preparation of human leucocytes for SEM observation

“…We have recently reported on procedures pertaining to the preparation of human leucocytes for scanning electron microscopy (SEM) observation (Abugaber et al, 1981 ;Lalague et al, 1982). The techniques utilized appeared to be efficient in yielding populations of spherical lymphocytes with finger-like, elongated and numerous microvilli.…”