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The Coolest Assay to Learn! Step-by-Step Guide to the Checkerboard Assay
- CLYTE research team
- Sep 15
- 6 min read
Are your two compounds a dynamic duo or a dysfunctional pair? You've tested Product A and Product B individually, and both show promise for increasing cell proliferation. But the million-dollar question remains: what happens when you combine them? Do they amplify each other's effects (synergy), simply work side-by-side (additivity), or worse, hinder one another (antagonism)?
Guesswork won't cut it in the lab. You need clear, quantitative answers. This is precisely where the Checkerboard Assay becomes an indispensable tool. It’s a robust and straightforward method for mapping the interactive landscape of two compounds across a gradient of concentrations. The result is a clear verdict on whether your combination is a breakthrough in the making or a dead end.
What Exactly is the Checkerboard Assay?
The checkerboard assay is a powerful in vitro technique that uses a two-dimensional matrix, typically set up in a 96-well plate, to test various concentration combinations of two substances simultaneously.
Axis 1 (e.g., Columns): A serial dilution of Compound A.
Axis 2 (e.g., Rows): A serial dilution of Compound B.
The Grid: Each well within the matrix contains a unique concentration pairing of Compound A and Compound B.
By measuring the biological response in each well (e.g., cell viability, proliferation, or inhibition) and comparing it to the expected effect if the compounds were merely additive, you can precisely quantify their interaction. This analysis is often performed using the Bliss Independence Model. The assay's versatility makes it a cornerstone in fields like antimicrobial testing, cancer research, and studies on drug/device combinations.
Checkerboard Assay Practical Protocol
Here is a detailed, day-by-day protocol for conducting a checkerboard assay to test the synergistic effect of two products (A and B) on the proliferation of dermal fibroblasts.
Day 1: Cell Seeding
The foundation of a successful assay is a healthy, consistent cell layer.
Prepare Cell Suspension: Create a cell suspension of dermal fibroblasts at a concentration of 7.5×104 cells/mL in 2% DMEM.
Seed the Plates: Dispense 100 µL of the cell suspension into the inner wells of two 96-well plates (providing technical replicates). This seeds approximately 7,500 cells per well.
Minimize Edge Effects: Fill the empty outer wells with sterile PBS or medium. This creates a moisture barrier, preventing evaporation from your experimental wells.
Incubate: Place the plates in an incubator at 37°C with 5% CO₂ for 24 hours. This allows the cells to attach and resume normal growth.
Day 2: Compound Addition & The Checkerboard Setup
This is where the matrix is created.
Confirm Confluency: Visually inspect the cells under a microscope to ensure they are approximately 70–80% confluent and appear healthy.
Prepare Dilutions: Create a series of 2-fold serial dilutions for both Product A and Product B in a serum-free medium (SFM).
Set Up the Plate Layout: Organize your plate as follows for a clear and logical matrix:
Columns 2–7: Serial dilutions of Product A (combined with Product B from rows).
Rows B–G: Serial dilutions of Product B (combined with Product A from columns).
Column 8: Product A alone (control for Product A's effect).
Column 9: Product B alone (control for Product B's effect).
Column 10: Negative Control (cells + SFM only, representing 100% viability).
Column 11: Positive Control (cells + a known toxic substance, e.g., water, for a kill control).
Blanks: Columns 1 & 12 and Rows A & H should contain medium only (no cells) to blank the plate reader.
Add Compounds:
For combination wells, add 100 µL of the appropriate Product A dilution and 100 µL of the Product B dilution.
For "A only" or "B only" wells, first add 100 µL of SFM, followed by 100 µL of the corresponding product dilution to maintain a consistent final volume.
Incubate Again: Return the plates to the incubator (37°C, 5% CO₂) for 48 hours to allow the compounds to take effect.
Day 3: The Readout - Viability Assay
It's time to measure the results. Using a metabolic indicator like AlamarBlue is a common method.
Prepare Reagent: Make a 10% AlamarBlue solution in a suitable medium.
Treat the Wells: Gently remove the treatment medium from all wells.
Add AlamarBlue: Add the AlamarBlue solution to every well, including the blanks.
Incubate: Incubate for 1 hours at 37°C & 5% CO₂, allowing viable cells to metabolize the dye.
Measure Fluorescence: Use a plate reader to measure the fluorescence at an excitation wavelength of 530–560 nm and an emission wavelength of 590 nm.
Data Analysis: Are Your Compounds Synergistic?
Raw fluorescence values don't tell the whole story. Here’s how to translate your data into a definitive answer using the Bliss Independence Model.
Normalize Data: Calculate the percentage of viability for each well relative to your negative control (NC), which is set to 100%.
Convert to Fractions: Divide the percentages by 100 to get fractional effects (e.g., a viability of 105% becomes a fractional effect of 1.05).
Calculate Expected Effect (Bliss Independence): The Bliss model predicts the combined effect if the two compounds act independently. The formula is:
Eexp = (A + B) − (A × B)
Where A and B are the fractional effects of each compound when used alone at that specific concentration.
Calculate the Bliss Score (ΔBliss): The difference between your observed result and the expected additive effect reveals the interaction.
ΔBliss = Eobs − Eexp
ΔBliss > 0 ⟹ Synergy (The combination is more effective than expected).
ΔBliss = 0 ⟹ Additivity (The combination effect is what was expected).
ΔBliss < 0 ⟹ Antagonism (The combination is less effective than expected).
Important Note: The interpretation of "effect" depends on your assay's goal. In our fibroblast proliferation example, a higher fractional value means more growth. Therefore, Synergy means the observed growth is greater than expected, and Antagonism means the observed growth is less than expected. In an antimicrobial or cancer assay where the goal is to kill cells, the "effect" would be the fraction of cells killed, and Synergy would mean the observed killing is greater than expected.
The formula stays the same, but the meaning of “effect” flips depending on whether we want growth or death.
Tips for a Successful Checkerboard Assay
Precision is Paramount: Small pipetting errors during serial dilutions can cascade and ruin the entire matrix. Be consistent and meticulous.
Master the Controls: Your controls are non-negotiable. Ensure each compound alone shows a clear dose-dependent response for the results to be valid.
Avoid Edge Effects: Always fill perimeter wells with PBS or medium.
Choose the Right Viability Assay: AlamarBlue and MTT are common; pick one that suits your cell type.
Check Solubility: If your compounds precipitate out of the solution when combined, it can falsely appear as antagonism. Always check for solubility issues.
Replicate Everything (Biological vs. technical replicates): Always run at least two technical replicates (two plates per experiment) and, ideally, multiple biological replicates to ensure your findings are robust and reproducible.
By following this guide, you can confidently use the checkerboard assay to move beyond speculation and generate hard data on how your compounds truly interact.
Why it's the coolest assay?
The checkerboard assay takes the guesswork out of combination testing. Instead of assuming A + B will help, it shows you whether they truly synergize, cancel each other, or simply add up. With careful setup, attention to dilution, and rigorous analysis, this assay can save you time, resources, and help you focus only on the combinations that matter.
Frequently Asked Questions (FAQ)
1. Do I always need AlamarBlue, or can I use MTT?
Both work. AlamarBlue is faster and non-destructive; MTT requires cell lysis but is very reliable.
2. How many dilutions of each compound should I use?
At least 6 two-fold dilutions are recommended, covering a wide concentration range.
3. Can I apply checkerboard to more than 2 products?
Not easily—3D matrices become complex. Stick to 2 compounds for clarity.
4. Why do we use the Bliss model?
It assumes independence between A and B. Other models exist (Loewe, Highest Single Agent), but Bliss is widely accepted for first-pass synergy.
5. How do I decide on the starting concentration?
Use the IC₅₀ or biologically relevant range for each compound, then work backward in dilutions.
6. Can the checkerboard assay be used with primary cells?
Yes, but expect more variability and lower tolerance to treatments compared to immortalized lines.