Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes.
This study investigated the effects of commonly used procedures for the isolation of leucocytes from human blood in comparison with cells in whole blood on the surface expression of CD11b and L-selectin (adhesion molecules which are known to be increased and decreased respectively by cell activation). Washing of granulocytes or monocytes with Hanks' buffered salt solution after separation by either dextran sedimentation or density gradient centrifugation, increased surface expression of CD11b (p < 0.05). The number of monocytes bearing CD11b was enhanced (p < 0.05) by dextran sedimentation and two layer density gradient centrifugation (Histopaque). The increase in CD11b expression on granulocytes was associated with enhanced binding of the cells to endothelial monolayers that were either untreated (r = 0.902; p < 0.001) or treated with tumour necrosis factor alpha (TNF-alpha) (r = 0.68; p = 0.004). The expression of L-selectin was reduced on granulocytes that had been isolated by dextran sedimentation followed by hypotonic lysis of contaminating erythrocytes. All isolates of granulocytes demonstrated a loss of L-selectin following activation with fMLP though this effect was less marked with cells subjected to erythrocyte lysis. The various separation methods had little effect on expression or distribution of CD11b or L-selectin on lymphocytes. We conclude that isolation of lymphocytes by density gradient centrifugation and of granulocytes and monocytes by dextran-sedimentation and centrifugation using Histopaque gradients, but avoiding washing and the use of hypotonic erythrocyte lysis, are appropriate techniques for studying the expression and function of adhesion molecules.
Macey
M G
MG
Department of Haematology, Royal London Hospital, Whitechapel, UK.
McCarthy
D A
DA
Vordermeier
S
S
Newland
A C
AC
Brown
K A
KA
D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D004730:Endothelium, Vascular / Q000166:cytology; D006098:Granulocytes / Q000166:cytology*; D006801:Humans; D066298:In Vitro Techniques; D019041:L-Selectin; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000166:cytology*; D016177:Macrophage-1 Antigen / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology
D015815:Cell Adhesion Molecules; D016177:Macrophage-1 Antigen; D019041:L-Selectin; D009240:N-Formylmethionine Leucyl-Phenylalanine