The fate of monocytes during 24 h of culture as revealed by flow cytometry and electron microscopy.
Culturing elutriation-purified and cryopreserved human monocytes gives a cell loss of about 60% after a week. The main loss is during the first 24 h when one cell population dies by apoptosis and secondary necrosis, while another survives with minimal signs of apoptosis and necrosis. We have studied this initial cell loss using flow cytometry (FCM) and electron microscopy (EM) in parallel. Thawed cells were cultured in ultra low attachment wells and studied by FCM using Annexin V, Propidium iodide (PI), JC-1, APO2.7 and APO-BrDU. The EM studies comprised both transmission EM (TEM) and scanning EM (SEM), the latter employing cells labelled with antiCD14/gold and Annexin V/gold. Cells were counted by light microscopy to provide cell recoveries. DNA ladder patterns were investigated by electrophoresis. Camptothecin (CAM) was used as an apoptosis inducer. In the first 6 h of culture, there was an apoptotic phase with Annexin V(+)/PI(-) positive cells in FCM, chromatin condensation in TEM, a rapid and short phase with Annexin V/gold positively labelled cells in SEM and the cells disappeared by 6 h. All of these effects were enhanced by CAM. The necrotic phase (6-24 h) was associated with Annexin V(+)/PI(+) in FCM, and the data at 24 h was in agreement with the semiquantitatvive results from TEM. Discrepancies in the results for CD14 and Annexin V between FCM and SEM indicated phagocytosis. APO2.7 and APO-BrDU increases also indicated an accumulation of ingested material in vital cells. Centrifugation of supernatants, labelling pellets with Annexin V/FITC and examination by flow cytometry revealed no Annexin V positive cell fragments. We found evidence of rapid and efficient phagocytosis. CAM not only induced apoptosis, but also appeared to stabilise the cell membrane and increase both cell recovery and phagocytosis.
Lund
Per Kr
PK
The Research and Development Group, Department of Clinical Chemistry, Ullevaal University Hospital, N-0407 Oslo, Norway. perlund@online.no
Namork
Ellen
E
Brorson
Sverre Henning
SH
Westvik
Ase-Brit
AB
Joø
Gun Britt
GB
Øvstebø
Reidun
R
Kierulf
Peter
P
D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008564:Membrane Potentials; D008854:Microscopy, Electron / Q000379:methods; D008928:Mitochondria / Q000502:physiology; D009000:Monocytes / Q000166:cytology*; D013997:Time Factors
D017304:Annexin A5; D018950:Lipopolysaccharide Receptors