Automatic Cell Deposition Unit

 

Single cell cloning with the BD FACSAria

The cell sorter at ICMS is fitted with a robotic arm that allows the single cell cloning of required events, this is more proficient than cloning by liming dilutions as the FACSAria takes approximately 3 minutes to clone single cells into a 96 well plate. The Aria can also clone into other tissue culture plates in a sterile manner e.g. 6, 24, 48 and 384 well plates.

The software also allows the operator to put a required number of cells into a well then sort a different population of cells in the next well. In this sort mode only a single population of cells can be sorted at the same time. The fucntion of Index sorting creates a mini-file to show which single cell went into which well. Confirmation that a single cell was been sorted can be confirmed by sorting flow cytometry beads made up in a Horse Raish Peroxidase (Sigma)/PBS solution (1.5 mg/ml for an Aria IIIu cell sorter) into 100 ul TMB High sensitivity substrate solution (BioLegend) in each 96 well plate. After sorting a single bead into each well the TMB solution turns blue after 15 min in the dark, see figure.

The robotic arm also allows sorting onto microscope slides.

Publication.

P Sabbattini, M Lundgren, A Georgiou, C Chow, G Warnes, N Dillion. Binding of Ikaros to the lambda5 promoter silences transcription through a mechanism that does not require heterochromatin formation. EMBO J. 20(11), 2812-22, 2001.

OR Rodrigues, S Monard, A rapid method to verify single-cell deposition setup for cell sorters. Cytometry A 89A:594-600, 2016