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Dye Drop Experimental Protocol

Dye drop methods use a sequence of solutions each made slightly denser than the last by addition of iodixanol (OptiPrepTM), an inert liquid used in radiology. Denser solutions displace the previous solution with high efficiency and minimal mixing. This effectively eliminates the need for mix and wash steps, where most of the uneven cell loss occurs.

On this page, we give an overview of the dye drop and deep dye drop protocols. For more detailed protocols used to generate our published results, please visit protocols.io.

Dye Drop

Dye drop experiment overview

Step 1: Start with adherent cells in growth medium

Step 2: Stain

Using a multi-channel pipette, gently add Hoechst+LIVE/DEAD Far Red Fluorescent dye (LDR) in 10% OptiPrepTM in phosphate buffered saline (PBS) along the wall of the wells.

Only dead cells are stained by LDR.

Step 3: Fix

Add 4% formaldehyde in 20% OptiPrepTM in PBS along the wall of the wells. This solution will displace the stain solution at the bottom of the well.

Step 4: Aspirate

Remove the solutions in the wells, leaving some formaldehyde at the bottom to minimize cell loss. Refill the wells with PBS, seal the plates.

Step 5: Image or Store

Image plates immediately or store fixed cells in PBS at 4°C up to several weeks.

Deep Dye Drop

Deep dye drop experiment overview

Step 1: Start with adherent cells in growth medium

Step 2: LDR + EdU Stain

Using a multi-channel pipette, gently add LIVE/DEAD Far Red FLuorescent dye (LDR) and EdU (5-ethylnyl-2’-deoxyuridine) in 10% OptiPrepTM in PBS solution along the wall of the wells.

Only dead cells are stained by LDR.

Steps 3-5: Same as Dye Drop

Store in PBS at the end of Step 5

Step 6: Permeabilize

Add 0.5% Triton X-100 in 10% OptiPrepTM to permeabilize cell membranes.

Step 7: Click Label EdU

Label EdU with a fluorescent dye azide via Click chemistry in 20% OptiPrepTM

Only S-phase cells are stained by fluorescently-labeled EdU.

Step 8: Aspirate

Remove solutions from the wells via gentle aspiration.

Step 9: Block

Add blocking solution.

Step 10: pH3 Antibody

Stain with a conjugated antibody against phospho-histone H3 (pH3) in 10% OptiPrepTM

Only M-phase cells are stained by the pH3 antibody.