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5 R ESULTS
5.2 CHO repertoire, FACS data & fluorescence microscopy
In order to generate transient transfected CHO cells, expressing a specific human endothelial receptor on their surface, CHO cells were transfected with either pAc-GFP1-N1 or pEGFP-N1 plasmid, containing one specific human endothelial receptor fused to the expression marker GFP. The receptor forms the extracellular part of the fusion protein whereas the GFP is located in the cytoplasm of the cell without affecting the extracellular domain. CHO-pAc-GFP1-N1 (mock) control cell line was solely transfected with the GFP-bearing expression vector, lacking any receptor sequence, consequently synthesizing cytosolic GFP only. In total, 16 transgenic cell lines could be generated: CHO-WT, CHO-GFP, CHO-CD36, CHO-ICAM-1, CHO-CD9, CHO-CD151, CHO-CD62-E, CHO-CD62-P, CHO-VCAM1, CHO-HRH1, CHO-MDR1, CHO-TNFR1, CHO-TNFR2, CHO-CD37, CHO-CD55 and CHO-CD81.
To ensure that all CHO cell lines were transfected successfully and that human endothelial receptors are expressed and detectable by specific antibodies on their surface, a flow cytometric analysis was performed, following the general gating strategy, mentioned above. Furthermore, the fluorescence pattern of the cytosolic marker protein GFP was investigated by fluorescence microscopy with living cells. In the following, Figure 13.1 and Figure 13.2 show the individual microscopic images as well as the FACS results of the transgenic CHO cell lines.
The percentages of particular cell populations, compared to the previous gate, are given in any section. Thereby, the percentage of GFP single positive cells include the APC or PE antibody stained and therefore double positive cell population. In the shown graphic, the black labels comprise the CHO-WT and CHO-GFP cell lines, serving as negative controls. The CHO-WT cell line does not express any marker fluorescence and could not be stained with the specifically used antibodies. Thereby, CHO-WT cells appear only within the double negative gates, providing the general baseline for further gating and sort approaches. The CHO-GFP cell line only expresses the cytosolic marker protein GFP (89.1% GFP single positive), thus appearing within the GFP positive gates, while remains negative for receptor specific APC or PE staining. The white labels indicate transgenic CHO cell lines, expressing a specific human endothelial receptor on their surface, known to bind P. falciparum infected erythrocytes.
Figure 13.1 (to be continued). Fluorescence analysis and FACS analysis of transgenic CHO cells, expressing human endothelial receptors.
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RESULTS
Figure 13.2 (continued). Fluorescence analysis and FACS analysis of transgenic CHO cells, expressing human endothelial receptors. The black labels indicate the CHO cell lines, serving as negative controls: non-transfected WT cells and mock-transfected (GFP) cell. The white labels indicate transgenic CHO cell lines, expressing a human endothelial receptor on their surface, known to bind P. falciparum infected erythrocytes. Those cell lines were used in several experimental setups. The 6 blue and green labels indicate transgenic CHO cell lines, expressing
MDR1TNFR1TNFR2CD37CD55CD81CX3CL1CD44
GFP channel merged BF channel GFP & APC / PEP4 / 5 double positive
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EPOR
within this thesis. The grey labels indicate transgenic CHO cell, expressing potential IE binding receptors, while the final establishment of the cell lines failed. The left section shows individual fluorescence images, recorded with EVOS FLauto fluorescence microscope at a 40x magnification. Green fluorescence, recorded via the GFP channel indicates the expression of cytosolic marker protein GFP in a distinct pattern. In the brightfield (BF) channel, cells were recorded with transmitted light. Both images were merged subsequently. Scale bar: 50 µm. The right section shows the results of the FACS analysis of the transgenic CHO cell lines, following the general gating strategy. Unstained GFP expressing cells are plotted in green, while APC and PE surface stained cells are plotted in red, as stated on the right side of the graph. The left section shows the individual microscopic images, taken by the EVOS FLauto fluorescence microscope, with a 40x magnification. Green fluorescence, recorded via the GFP channel indicates the expression of cytosolic marker protein GFP in a distinct pattern. In the brightfield (BF) channel, cells were recorded with transmitted light. Both channels were recorded separately and images were merged together subsequently, with a scale bar of 50 µm. In the right section, the FACS gates show the actual cell population via an overlay view that merges the same individual gate of the respective cell line. Unstained CHO cells are plotted together with specifically stained CHO cells of the same parental population. Here, the grey color indicates unstained CHO-WT cell populations with no fluorescence exhibited. The green color indicates unstained CHO cells, expressing cytosolic GFP only. The red color indicates CHO cell populations, expressing a specific human endothelial receptor on their surface, additionally stained with the red appearing APC or PE antibody, respectively.
The white label comprising CHO-CD36 (91.3% GFP and 99.7% GFP & APC positive), CHO-ICAM-1 (76.8% GFP and 99.9% GFP & APC positive), CHO-CD9 (99.8% GFP and 100% GFP & PE positive), CHO-CD151 (88.8% GFP and 96.6% GFP & APC positive), CHO-CD62-E (92.1% GFP & 86.9% GFP & APC positive), CD62-P (67.8% GFP and 88.7%
GFP & PE), CHO-HRH1 (80.0% and 70.9% GFP & PE) and CHO-VCAM1 (98.4% GFP and 99.2% GFP & PE) cell lines, used in several experimental setups subsequently. The grey labels combine the transgenic cell lines CHO-EPOR (0.55% GFP & 7.41% GFP & APC), CHO-CX3CL1 (2.83% GFP and 33.3% GFP & APC) and CHO-CD44 (89.6% GFP and 2.97%
GFP & APC), expressing potential IE binding receptors, while the final establishment of the cell lines failed. Eventually, it was either not possible to detect a sufficient number of cells, expressing GFP, or detected cells lacking specific surface antibody staining with APC or PE respectively. However, the transgenic cell lines CHO-MDR1 (72.1% GFP and 98.3% GFP & PE), CHO-TNFR1 (96.3% GFP and 99.8% GFP & PE), CHO-TNFR2 (99.8%
GFP and 99.9% GFP & APC) belong to the dark blue label, are also known to bind P. falciparum IE and were particularly focused within this thesis. Furthermore, the light blue label includes CHO-CD37 (25.4% GFP and 64.1% GFP & APC), CHO-CD55 (39.1%
GFP and 100% GFP & APC) and CHO-CD81 (88.6% GFP and 100% GFP & APC) cell lines, potentially able to bind P. falciparum IE in a pertinent manner. Also, those cells lines were especially focused within this thesis.
> Taken together, it was possible to establish 16 out of 19 transgenic CHO cell lines with a reliable level of detectable human endothelial receptor protein expression on the cell surface.
RESULTS