Fig. 8
Single-cell sub-cloning procedures of genetically modified cells, GFP expression and cell viability in subcloned cells. (A) Precise microliter liquid intake performance trial studies with pipette system for CRISPR.BOT V2. (B) Seeding of Cells. CRISPR + gRNA 1-2-3 Jurkat Cells (Clone E6-1, TIB-152, ATCC) were cultivated at 5 × 104 cells/well in a 6-well. (C) Cell dilution. 10 µl of 3 different viruses in the wells of the 6-well plate were taken separately. 1 ml/500 cells were taken from the cell + RPMI mixture with a total volume of 1 ml and added onto 9 ml RMPI medium and placed in 6-well. (D) Integration of plates into the autonomous robot system. 6-well and 96-well plates were placed on the Robot’s frame. (E) Sub-cloning of cells into 96-well plate. CRISPR + gRNA 1-2-3 Jurkat Cells (Clone E6-1, TIB-152, ATCC) in the 6-well plate were pulled at 100° and added to a whole 96-well plate. (F) Incubation of the 96-Well plate. (G) CRISPR.BOT-based subcloning of the transgenic cells. (H) Shows the positive and negative rates of GFP expression in cells sorted in the experiment performed with CRISPR.BOT. The positive rate of GFP expression (more than 90%) is defined as “GFPhi”, while the negative rate (less than 10%) is called “GFPlo”. At the end of the experiment, the majority of the samples were sorted, resulting in a statistically significant result. Furthermore, some samples showed moderate GFP expression (between 10% and 90%), which is referred to as “GFPint”. (I) Shows the percentage of viability of subcloned cells and indicates that the statistical significance of these results was confirmed by unpaired t-test, *p < 0.05. Not significant, NS. µl, microliter; GFP green fluorescent protein, gRNA guide RNA. This indicates that the results are reliable and that the experiment was successful.
