Figure 5.1: The effect of used substrate and medium on the proportion of Young Clone 8+ cells adhered to the substrate 24 hours after initiation
The mean proportion of YCl.8+ cells adhered to the O.Cl.8+ substrate has dropped from over 80% to less than 25%. More YCl.8+ cells adhered to the Old used substrate with fresh medium and Young used medium than Old used medium.
Figure 5.2: The effect of used substrate and medium on the proportion of Old Clone 8+ cells adhered to the substrate 24 hours after initiation. The mean proportion of OCl.8+ cells adhered to the Y.Cl.8+ substrate has almost doubled from less than 25% to more than 40%. More OCl.8+ cells adhered to the Young used substrate with fresh medium and Young used medium than Old used medium.
Figure 5a:YCl.8+ and Cl.8R cell morphologies 24 hours after initiation.
DIC images of Young Clone 8+ and Clone 8R cells showing elongate, round, macrophage and teardrop shaped cells which are found in these cultures 24 hours after initiation. The prospective macrophage cell types have already flattened and are putting out cell processes.
e, elongate cell type, f, flattened cell process in teardrop shaped cell, m, macrophage cell type, r, round cells. Scale bar represents:20μm
Figure 5b: Low passage Clone 8+ cells 24 hours after initiation.
Phase contrast image of low passage Clone 8+ cells 24 hours after initiation. Cells are already beginning to show signs of migration and aggregation. The elongate cells are in the majority with fewer round cells in evidence. a, aggregate; r, round cell
Scale bar represents:50μm
Figure 5c: Low passage Clone 8+ cells day 4 after initiation Phase contrast image of low passage Clone 8+ cells 24 hours after initiation which shows the substantial
aggregation of cells observed in these cultures. Migration of cells has left areas of denuded substrate. Cells within the aggregates are close together and on top of one another. These cells have assumed a spherical morphology.
a, aggregate
Figure 5d(i): Low passage Clone 8+ cells day 6, confluence.
Phase contrast image of low passage Clone 8+ cells showing the characteristic pattern of aggregation and association of single cells normally associated with the low passage Cl8+ cells, YCl.8+ and Cl.8R. The phase bright aggregates contain cells which are closely associated, individual cells are hard to distinguish. a, aggregates; nc, area of substrate where no cells are present.
Scale bar represents:50μm
Figure 5d(ii):Aggregates connected by single cells in Low passage Clone 8+
Phase contrast image of low passage Clone 8+ cells showing elongate phase dark single cells with long processes connecting the aggregates which are phase bright, it at this stage that routine passaging of the cultures takes place.
ec, elongate cell
Scale bar represents:20μm
Figure 5e: High passage Clone 8+ cells 24 hours after initiation.
Phase contrast image of high passage Clone 8+ cells 24 hours after initiation which shows that the majority of cells present in these cultures were phase bright with a spherical morphology. The spherical cells were only loosely attached to the substrate and appeared loosely associated with each other. Phase dark elongate cells were also present attached to the substrate.
a, aggregate; e, elongate cells; s, spherical cells Scale bar represents:50μm
Figure 5f:High passage Clone 8+ cells 3 days after initiation
DIC image of low passage Clone 8+ cells 3 days after initiation showing the density of cells suspended in these cultures. The substrate beneath cannot be visualised due to the number of cells suspended in the medium, a number of phase bright areas in the cell aggregations can be seen.
p, phase bright cell aggregations Scale bar represents:50μm
Figure 5g:Low passage Clone 8+ cells 24 hours after ecdysone
DIC image of low passage Clone 8+ cells 24 hours after ecdysone which shows the flattened appearance
assumed by the cells after ecdysone. Many cell processes extending from the cells can be seen and cells have spread out onto the substrate.
cs, cell spreading; p, processes Scale bar represents:20μm
Figure 5h:High passage Clone 8+ cells 24 hours after ecdysone
DIC image of high passage Clone 8+ cells 24 hours after ecdysone showing cells suspended in the medium. Single cells with a flatter appearance and much longer processes extending into the medium can be seen. These processes appear to be associating with spherical cells. p, processes; pc, processes which are in contact with the spherical cells
Scale bar represents:20μm
Figure 5i: Low passage YCl8+ cells on used Old Cl8+ substrate, day 2
Phase contrast image showing the disorganised way in which YCl8+ cells appeared to grow when compared to the control, figure 5b. Few elongate cells were present in these cultures and cell aggregation and migration was reduced. Phase bright aggregations were only loosely attached to the substrate. Cells firmly adhered to the substrate have assumed a phase dark appearance. a, aggregations loosely attached; c, cells firmly attached to the substrate
Scale bar represents:50μm
Figure 5j:Low passage YCl.8+ cells on used OCl.8+ substrate, day 3.
Phase contrast image of the YCl8+ cells, cell aggregation is apparent and there are greater numbers of elongate cell present. Migration of cells is still reduced, more cells can be seen adhered to the substrate between the
aggregations.
a, aggregate suspended in medium; c, phase dark cells firmly adhered to the substrate; e, elongate cell
Figure 5k:Low passage Old Cl.8+ on used Young Cl.8+ substrate, day 2
Phase contrast image of OCl.8+ cells showing cell aggregations which may only be loosely attached to the substrate. Phase dark, elongate and round cells cells were present in these cultures.
a, aggregates; p, phase dark cells Scale bar represents:100μm
Figure 5m: Old Cl.8+ on used Young Cl.8+ substrate, day 5
Phase contrast image of the tight aggregations which had formed in these cultures which on appearance were loosely attached to the substrate. A number of phase dark cells can be seen adhered to the substrate lying beneath the aggregates and elongate cells were observed although infrequently. No cells grew in many areas of the substrate in these cultures.
a, aggregate; e, elongate cell type; p, phase dark cells; s, substrate.