To obtain an even monolayer, mix cell suspensions well before adding them into the plate. It may be necessary to agitate the reservoir to ensure the best suspension possible. Add cells gently using a multichannel pipette with the plate on a flat surface. Once cells are plated, leave the plate undisturbed for 10 min at room temperature to ensure an even monolayer adheres to the plastic.
To avoid disturbing the monolayer when adding effector cells, dribble cells down the edge of the well, angling the pipette tip towards the edge of the well.
To reduce costs, target cell survival can be tracked using Nuclight Red fluorescence alone (without an indicator of apoptosis) since these cells lose fluorescence when they die.
Important controls include target alone wells, targets with drugs and no effectors, targets with effectors and no drugs.
Variation between replicates wells
Diagnosis: a large standard deviation in live targets per well in the first image. Inconsistent increase in image confluence between the first image and when effectors were added.
Fix: If target cell variation is a problem increase the number of replicate wells until technique improves. For effector cell variation, a 96 well 'master plate' containing all effectors and drugs to be added will reduce the time between the first effectors being added and the last effectors being added to the target cells across multiple conditions.
Condensation, air bubbles or marking on the plastic impair target cell counts.
Diagnosis: single replicates have sudden changes in target cell number or blurry images are apparent in initial frames.
Fix: Bubbles in the well, condensation on the lid or markings on the plastic within the light path will reduce the quality of the images. Bubbles can be popped using a wash bottle containing 70% ethanol with the tube removed to prevent ethanol liquid leaving the bottle. Ethanol vapors will pop the bottle when gently blown onto the meniscus layer. Do not mark plates and pre-warm them in the incubator for 15 min prior to imaging to avoid condensation and marks on the plastic impairing the images.
Effector cells are added too harshly and disturb the target cell monolayer.
Diagnosis: target cell counts drop dramatically between first image and the addition of effectors with distinct zones of the image that are clearly effected.
Fix: check that poly-L-ornithine is working effectively. Alter technique for addition of effectors. Normalize to the first image after effectors are added to avoid spuriously implying effector cells are killing target cells that have been knocked out of the field of view.
Tumor alone condition becomes confluent impacting the apparent rate of cytotoxicity normalized to tumor alone controls.
Diagnosis: target cell numbers in control wells plateau.
Fix: reduce the number of target cells per well to ensure they reach confluence at the end of the full assay, not before.
Diagnosis: target cell numbers decrease over time in the tumor alone condition.
Fix: Drop the threshold of detection for target cell masks if using amine reactive dyes to ensure target cells can be detected over time after they have divided. Size thresholds on the overlap masks (target cells+ caspase 3/7+) will ensure small dead effectors (caspase 3/7+) floating above live target cells do not get counted as dead target cells.