Review and General Details of the Techniques Employed to Prepare Leukocytes

Abstract

Leukocytes were generally prepared from buffy coats obtained from normal heparinized venous blood by centrifugation for 5–8 min at 800–1000 rpm. Leukocyte fractions were obtained using the Ficoll-Hypaque density gradient centrifugation technique [11], which separates erythrocytes and granulocytes from mononuclear cells (lymphocytes and monocytes). This technique allows one to harvest granulocytes and mononuclear cells separately. Mononuclear cell suspensions contain mainly small lymphocytes with varying numbers of monocytes, while the percentage of contaminating polymorphonuclear leukocytes varies ranging from 0–5%. Cultured cells were concentrated, like circulating leukocytes, by gentle centrifugation at 800–1000 rpm for 5–8 min. This was followed by resuspension of the cell pellet in the original culture medium or directly in glutaraldehyde fixative or by fixation after sedimentation within the culture flask in situ.