Example data table
| Condition | Replicates | Mean | Notes |
|---|---|---|---|
| Blank | 0.02 | 0.02 | Media/background |
| Spontaneous | 0.12, 0.11, 0.13 | 0.12 | Untreated cells |
| Maximum | 0.95, 0.98, 1.02 | 0.98 | Lysis control |
| Sample | 0.45, 0.43, 0.47 | 0.45 | Treated condition |
Using LDH mode with blank 0.02, this example yields a mid-range cytotoxicity index.
Notes and interpretation
- Values below 0% or above 100% can occur from noise or control drift.
- For LDH, ensure maximum control is clearly higher than spontaneous.
- For viability signals, confirm the control represents healthy baseline.
- Use consistent plate layouts and timing to reduce variability.
Why cytotoxicity indexing matters
Cytotoxicity measurements turn complex cell responses into a comparable percentage tracked across plates, days, and operators. By normalizing a sample signal between baseline and a defined extreme, the index reduces dependence on absolute absorbance. This supports dose-response screening, formulation comparisons, and batch release decisions. A single index is easier to share in reports while preserving replicate details for traceability. It also enables quick triage of compounds before deeper mechanistic assays are run for researchers.
Selecting the right control strategy
LDH release assays use spontaneous leakage as the low anchor and chemically lysed wells as the high anchor. Viability assays treat the untreated control as the viable baseline and convert reduced signal into cytotoxicity. A blank well can subtract media and reagent background. Controls should be distributed across the plate to detect edge effects and prepared with the same timing as samples. Add vehicle controls when solvents could affect cells.
Replicates, variability, and quality flags
Replicates improve confidence because the mean is less sensitive to random noise and pipetting error. This calculator reports means and standard deviations, then estimates variability using coefficient of variation checks. High CV suggests inconsistent dispensing, uneven cell seeding, or bubbles. Outlier wells can be rechecked visually before exclusion. When controls are weakly separated, small denominators amplify noise; the quality badge highlights when results deserve confirmation.
Interpreting values outside 0–100%
Negative values occur when treated wells read below spontaneous leakage or above the untreated control, due to assay scatter. Values above 100% appear if the maximum control is underestimated, saturation occurs, or the sample interferes. Instead of clipping, keep the raw output, review control integrity, and rerun with improved lysis conditions or dilution to stay in the linear range. Confirm with parallel orthogonal readouts.
Reporting and reproducibility workflow
For documentation, record assay mode, blank setting, replicate counts, and the formula used. Export the CSV for analysis and the PDF for notebooks. Note instrument wavelength, incubation time, and any normalization. When comparing experiments, keep control definitions constant and use the same replicate structure so indices remain comparable. Pair the index with morphology checks or apoptosis markers when mechanisms matter. Store exports with sample identifiers.
FAQs
1) What does a cytotoxicity index of 50% mean?
It means the sample signal sits halfway between baseline and the defined high control in your selected mode, suggesting moderate toxicity under those assay conditions.
2) Why do I get negative cytotoxicity values?
Negative results usually come from normal measurement noise, plate effects, or a sample reading slightly above the control (viability mode) or below spontaneous (LDH mode). Recheck controls and replicate consistency.
3) Can I use different numbers of replicates?
Yes. Enter any count of replicate readings per field. More replicates generally reduce uncertainty and improve the stability of the mean and variability checks.
4) Should I always subtract a blank?
Use a blank when media, reagent, or plate background meaningfully contributes to signal. If your instrument is already blanked or background is negligible, leave it at zero.
5) What if my maximum control is lower than spontaneous?
That indicates a setup problem, such as incomplete lysis, timing mismatch, or pipetting error. Fix the control procedure before interpreting sample indices.
6) Which mode should I choose for MTT or resazurin?
Select viability mode because higher signal reflects healthier cells. The calculator converts the sample-to-control ratio into cytotoxicity percentage after optional blank subtraction.