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Cell lines were methanol fixed and permeabilised prior to incubation with Geminin-AF488 antibodies and DAPI staining. Acquisition, analysis and sorting was performed on the BD FACSMelody using the BD FACSChorus™ software. Analysis was performed using Flow Jo v10.10. PEO1 Clone B4 used to demonstrate gating. The gating tree was as follows: a ) FSC-A/SSC-A (gating of cells based on size and intracellular composition) b ) SSC-H/SSC-W and c ) FSC-H/FSC-W for removal of non-singlets d ) DAPI-A/Geminin-AF488-A for cell cycle analysis. Geminin is a DNA replication regulator expressed in S and G2 phase of the cell cycle, and not expressed in G1-phase of the cell cycle. DAPI is used as a DNA content marker, and is routinely used in cell-cycle analysis. Together, geminin and DAPI intensity are used here to gate cells in G1-phase of the cell cycle. The use of a primary-antibody negative control population (AF488 and DAPI staining only) was used to determine the gating of cells with negative expression of geminin. The geminin-negative population were sorted via FACS and subsequent single cell isolation was performed using the <t>cellenONE®</t> cell dispenser.
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Cell lines were methanol fixed and permeabilised prior to incubation with Geminin-AF488 antibodies and DAPI staining. Acquisition, analysis and sorting was performed on the BD FACSMelody using the BD FACSChorus™ software. Analysis was performed using Flow Jo v10.10. PEO1 Clone B4 used to demonstrate gating. The gating tree was as follows: a ) FSC-A/SSC-A (gating of cells based on size and intracellular composition) b ) SSC-H/SSC-W and c ) FSC-H/FSC-W for removal of non-singlets d ) DAPI-A/Geminin-AF488-A for cell cycle analysis. Geminin is a DNA replication regulator expressed in S and G2 phase of the cell cycle, and not expressed in G1-phase of the cell cycle. DAPI is used as a DNA content marker, and is routinely used in cell-cycle analysis. Together, geminin and DAPI intensity are used here to gate cells in G1-phase of the cell cycle. The use of a primary-antibody negative control population (AF488 and DAPI staining only) was used to determine the gating of cells with negative expression of geminin. The geminin-negative population were sorted via FACS and subsequent single cell isolation was performed using the <t>cellenONE®</t> cell dispenser.
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Cell lines were methanol fixed and permeabilised prior to incubation with Geminin-AF488 antibodies and DAPI staining. Acquisition, analysis and sorting was performed on the BD FACSMelody using the BD FACSChorus™ software. Analysis was performed using Flow Jo v10.10. PEO1 Clone B4 used to demonstrate gating. The gating tree was as follows: a ) FSC-A/SSC-A (gating of cells based on size and intracellular composition) b ) SSC-H/SSC-W and c ) FSC-H/FSC-W for removal of non-singlets d ) DAPI-A/Geminin-AF488-A for cell cycle analysis. Geminin is a DNA replication regulator expressed in S and G2 phase of the cell cycle, and not expressed in G1-phase of the cell cycle. DAPI is used as a DNA content marker, and is routinely used in cell-cycle analysis. Together, geminin and DAPI intensity are used here to gate cells in G1-phase of the cell cycle. The use of a primary-antibody negative control population (AF488 and DAPI staining only) was used to determine the gating of cells with negative expression of geminin. The geminin-negative population were sorted via FACS and subsequent single cell isolation was performed using the <t>cellenONE®</t> cell dispenser.
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Cell lines were methanol fixed and permeabilised prior to incubation with Geminin-AF488 antibodies and DAPI staining. Acquisition, analysis and sorting was performed on the BD FACSMelody using the BD FACSChorus™ software. Analysis was performed using Flow Jo v10.10. PEO1 Clone B4 used to demonstrate gating. The gating tree was as follows: a ) FSC-A/SSC-A (gating of cells based on size and intracellular composition) b ) SSC-H/SSC-W and c ) FSC-H/FSC-W for removal of non-singlets d ) DAPI-A/Geminin-AF488-A for cell cycle analysis. Geminin is a DNA replication regulator expressed in S and G2 phase of the cell cycle, and not expressed in G1-phase of the cell cycle. DAPI is used as a DNA content marker, and is routinely used in cell-cycle analysis. Together, geminin and DAPI intensity are used here to gate cells in G1-phase of the cell cycle. The use of a primary-antibody negative control population (AF488 and DAPI staining only) was used to determine the gating of cells with negative expression of geminin. The geminin-negative population were sorted via FACS and subsequent single cell isolation was performed using the cellenONE® cell dispenser.

Journal: bioRxiv

Article Title: scUnique: Measuring Ongoing Chromosomal Instability

doi: 10.64898/2026.04.28.721240

Figure Lengend Snippet: Cell lines were methanol fixed and permeabilised prior to incubation with Geminin-AF488 antibodies and DAPI staining. Acquisition, analysis and sorting was performed on the BD FACSMelody using the BD FACSChorus™ software. Analysis was performed using Flow Jo v10.10. PEO1 Clone B4 used to demonstrate gating. The gating tree was as follows: a ) FSC-A/SSC-A (gating of cells based on size and intracellular composition) b ) SSC-H/SSC-W and c ) FSC-H/FSC-W for removal of non-singlets d ) DAPI-A/Geminin-AF488-A for cell cycle analysis. Geminin is a DNA replication regulator expressed in S and G2 phase of the cell cycle, and not expressed in G1-phase of the cell cycle. DAPI is used as a DNA content marker, and is routinely used in cell-cycle analysis. Together, geminin and DAPI intensity are used here to gate cells in G1-phase of the cell cycle. The use of a primary-antibody negative control population (AF488 and DAPI staining only) was used to determine the gating of cells with negative expression of geminin. The geminin-negative population were sorted via FACS and subsequent single cell isolation was performed using the cellenONE® cell dispenser.

Article Snippet: PEO1 single-cell clones were generated by sorting single cells into 96-well plates containing complete media using the cellenONE® cell dispenser (Scienion).

Techniques: Incubation, Staining, Software, Cell Cycle Assay, Marker, Negative Control, Expressing, Single-cell Isolation, Cellenone

Journal: bioRxiv

Article Title: scUnique: Measuring Ongoing Chromosomal Instability

doi: 10.64898/2026.04.28.721240

Figure Lengend Snippet:

Article Snippet: PEO1 single-cell clones were generated by sorting single cells into 96-well plates containing complete media using the cellenONE® cell dispenser (Scienion).

Techniques: Cellenone, Isolation, Transmission Assay, Selection