# Single-Cell Patterning Based on Immunocapture and a Surface Modified Substrate
## 3 Single Cell Patterning
### Results and Discussions
#### 3.3.2 HL-60 Cell Seeding Results  
(1) Selection of antibodies. The cell seeding result is shown in Figure 9, where the green fluorescence is the patterning result of the streptavidin. The cells were stained with red cell membrane marker DiIC18(3). The yellow fluorescence was the superposition effect of the red fluorescence and the green fluorescence. Owing to the strong binding ability between the streptavidin and biotins, CD15 antibodies can be effectively patterned. The red dots are the fluorescence produced by the cells under excitation, and shows that the cells are in disorder. No cells are present, either on the arrayed patterns or on the bar frame with larger dimensions. The expression rate of CD15 antigens on the surface of the HL-60 cells was investigated, and the experimental results show that this antigen is low expressed on the HL 60 cells, that is, only approximately 22% (Supplementary materials Figure S2). To obtain good single-cell patterns, it is necessary to use antigens that are high expressed on the surface of the HL-60 cells.HL-60 is a promyelocytic leukemia cell with immaturity and is easy to be differentiated under the influence of various environment factors, thus leading to the change in cell-membrane-expressed antigens. In such cases, only suitable typical expressed antigens can guarantee the expression in the differentiated HL-60 cell lines. CD45 is a typical antigen of leucocyte, which is widely found on the surface of white blood cells. Therefore, we chose biotinylated CD45 antibodies as the direct binding proteins for the cells seeding. To test the binding effect of CD45, the same experiment was conducted with CD45 on a substrate with no features, indicating good capture results (Supplementary materials Figure S3). Figure 8: Streptavidin patterning results. The largest diameter is 72 \(\upmu\)m and the smallest diameter is 8.4 \(\upmu\)m. It can be seen that the Streptavidin patterning results is very good. (**a**) Patterns with big diameters. (**b**) Patterns with smallest diameters. (**c**) Patterns with largest diameters. (**d**) Patterns with middle diameters. 2) Cell concentration. Cell concentration will affect the subsequent cleaning process and the patterning results. Real-time observation under a microscope indicated that when the cell concentration is extremely high, the cells will gather during incubation. Therefore, when removing the uncaptured cells with the subsequent cleaning process, the captured cells will likely be rinsed, thus leading to pattern defects. Meanwhile, when cell concentration is extremely low, the cells cannot cover the substrate surface, thus leading to vacancies in patterning. The diameter of the HL-60 cell is between 10 \(\upmu\)m and 12 \(\upmu\)m. Suppose that the cells are compactly spread over the substrate, and using 10 \(\upmu\)m as cells' diameter, the cells' density when covering 1 cm\({}^{2}\) area should be the following: (1 cm/10 \(\upmu\)m)\({}^{2}\)/1 cm\({}^{2}\) = 1 \(\times\) 10\({}^{6}\)/cm\({}^{2}\). According to experience, each layer of the cell in suspension generally occupies 2 mm in depth; subsequently, the cell concentration covering the whole surface should be 1 \(\times\) 10\({}^{6}\)/(2 mm \(\times\) 1 cm\({}^{2}\)) = 5 \(\times\) 10\({}^{6}\)/mL. In practice, the concentration in cell patterning could be slightly lower than the calculated value (Supplementary materials Figure S4). The mediums and buffers were all standard solutions. The experiments were conducted at room temperature (24-28 \({}^{\circ}\)C). There is no obvious difference in the binding ratio and yield. (3) Time of cell seeding  
The time of cell seeding is determined by the reaction time of the surface antigens and antibodies. If the seeding time is extremely short, the antigen-antibody reaction will be insufficient, thus leading to the failure of some pattern islands.