The application of flow cytophotometry in measurements of cell adhesion

Summary A common approach to the study of cell substrate interactions is the measurement of the attachment of cells to different substrates or to cultured cell layers. The evaluation of attachment is made either by scintillation counting of previously labelled adhering cells, or by light microscopy using the criterion of cell shape, sometimes refined by automatic image analysis. These methods have many drawbacks. This paper suggests the use of fluorescence-activated flow cytophotometry, (FC) which yields direct counts of the non-adhering cells. These free cells are removed after completion of the adhesion experiment from the microtitre plate wells. An internal standard, in the form of fluorescent polystyrene beads is added, allowing evaluation of the percentage of cells adhering to the well walls. Flow cytophotometry then produces data based on the analysis of large populations of cells. Unequivocal discrimination is obtained between the counted cells and counted fluorescent beads eliminating counting errors. The results can be processed on line by computer.A suspension of mouse splenocytes was used for the evaluation of the overall error of the method arising from maccuracies in pipetting, interference of glutaraldehyde with ethidium bromide (EB) staining and instrumental error. Each adhesion experiment was terminated by staining and post-fixation and it was established that this introduces no change in cell counting, in comparison with the original unfixed cells. Prefixation, however, quenches the EB staining and would interfere with the counting procedure. The overall standard error of the technique was found to be 5%–10%.A comparison was made of this technique with conventional cell counting by light microscopy, in a study of mouse splenocytes and of myoblasts isolated from 12 day old chicken embryos, Suspensions of these cells and of myoblasts were applied to microtitre plate wells precoated with fibronectin or Concanavalin A (Con A). The specific phase of cell contact was blocked by preincubation of the cells in media containing Con A or -methyl-d-mannoside, and the kinetics of cell attachment to Con A coated surfaces were compared. Claims in the literature for a difference between the fibronectin-type and lectin-type curves were not supported by our results. It was found that the curves for the attachment of both cell types to fibronectin or Con A coated surfaces are similar in shape.The results obtained by the FC technique were evaluated statistically and compared with cell counts obtained by microscopy. The definition of adhering cells is different in flow-cytophotometry (i.e. cells which cannot be removed from wells) from that used in microscopic counting (i.e. cells which have lost their round shape). Nevertheless, the results are found to be parallel which allows us to suggest the use of flowcytophotometry as a routine technique for evaluation of cell adhesion.