Zubair Khalid

Virologist/Molecular Biologist | Veterinarian | Bioinformatician

Conventional & Molecular Virology • Vaccine Development • Computational Biology

Dr. Zubair Khalid is a veterinarian and virologist specializing in conventional and molecular virology, vaccine development, and computational biology. Dedicated to advancing animal health through innovative research and multi-omics approaches.

Dr. Zubair Khalid - Veterinarian, Virologist, and Vaccine Development Researcher specializing in Computational Biology, Multi-omics, Animal Health, and Infectious Disease Research

Category: Guides

Cell Titer Glo Protocol: A Practical Guide for Cell Viability Assays

The CellTiter Glo assay is a homogeneous, luminescence based method for measuring adenosine triphosphate (ATP) as a direct indicator of metabolically active, viable cells. This guide explains the protocol step by step, including decision points, quality checks, and interpretation limits. Use this guide if you are a cell biologist, pharmacologist, or graduate student planning to evaluate cell proliferation, cytotoxicity, or compound efficacy in vitro. The assay is widely used in cancer research, virology, and drug development because it is sensitive, rapid, and compatible with multiwell plates. For a foundational overview of cell viability assays, consult the NCBI Bookshelf. Additional training materials on assay design and data analysis are available from EMBL EBI Training.

At a Glance

Feature Description
Purpose Quantify viable cells based on ATP content
Principle Luciferase reaction uses ATP from live cells to generate a stable luminescent signal
Format 96 well, 384 well, or 1536 well plates, adherent or suspension cells
Readout Luminescence (relative light units, RLU)
Total time 10 minutes after reagent addition (signal stable for 30 minutes)
Sensitivity Detects as few as 10 cells per well (depending on plate format)
Common applications IC50 determination, cell proliferation assays, cytotoxicity screening, viral replication inhibition studies

Decision Criteria

Before running the protocol, consider whether CellTiter Glo is the right assay for your question. The assay measures ATP, which correlates well with viable cell number under most conditions. However, when cells undergo metabolic stress without losing viability, ATP levels can shift independently of cell count. For example, compounds that inhibit mitochondrial function may lower ATP without killing cells. In such cases, a complementary assay (e.g., trypan blue exclusion or caspase activity) is advisable.

Choose the plate format based on cell type and throughput. 96 well plates are standard for initial optimization. 384 well plates are suitable for dose response curves. The recommended cell seeding density depends on the growth rate of your line. For most adherent cells, seed 1,000 to 10,000 cells per well in 100 µL of medium for a 96 well plate. Optimize density so that untreated control wells reach 70 to 80 percent confluence at the time of measurement. Include a medium only blank to subtract background luminescence. Also include a positive control known to reduce viability (e.g., 1 µM staurosporine) and a vehicle control (e.g., DMSO at the same final concentration). The clinical studies described in Caveolin 1 inhibits the proliferation and invasion of lung adenocarcinoma via EGFR degradation and Differential response of patient derived primary glioblastoma cells to metabolic and adhesion inhibitors used ATP based viability assays to evaluate therapeutic effects, demonstrating the assays utility in complex cell models.

Practical Workflow or Implementation Steps

Step 1: Prepare Cells

Harvest cells at log phase growth. Count viability with trypan blue and ensure greater than 90 percent viability. Resuspend in complete medium at the desired density. Dispense the cell suspension into a white, opaque walled multiwell plate. White plates minimize well to well cross talk and improve signal to noise. Allow adherent cells to attach for 4 to 24 hours depending on cell type before adding treatments.

Step 2: Apply Treatments

Add compounds or test conditions. For dose response studies, use serial dilutions in triplicate. The final DMSO concentration should remain below 0.5 percent (preferably 0.1 percent) to avoid solvent toxicity. Include untreated controls and a blank containing medium only. Incubate for the desired exposure period. For acute cytotoxicity, 24 to 72 hours is typical. For viral replication assays, measurement at 48 to 72 hours post infection is common, as shown in Measuring Influenza Virus Infection Using Bioluminescent Reporter Viruses.

Step 3: Equilibrate Plate

Remove the plate from the incubator and let it sit at room temperature for 30 minutes. This step ensures that the temperature of the plate and the CellTiter Glo reagent match, which is critical for consistent luciferase kinetics.

Step 4: Add CellTiter Glo Reagent

Thaw the CellTiter Glo reagent and mix gently. Add a volume of reagent equal to the volume of culture medium present in each well (e.g., 100 µL reagent to 100 µL medium). Use a multichannel pipette for 96 well plates or a reagent dispenser for 384 well plates to add quickly and consistently. Avoid introducing bubbles.

Step 5: Mix

Place the plate on an orbital shaker for 2 minutes at 300 to 500 rpm. Mixing is essential to lyse cells completely and release ATP. Ensure that the shaker speed does not cause spillover between wells.

Step 6: Incubate

Incubate the plate at room temperature for 10 minutes to allow the luminescent signal to stabilize. The signal is stable for up to 30 minutes, but read all plates within this window to minimize variability.

Step 7: Measure Luminescence

Read the plate on a luminometer with an integration time of 0.25 to 1 second per well. Record RLU values. Export data to a spreadsheet for analysis.

Quality Checks

Run a control plate with serial dilutions of cells to confirm linearity between cell number and RLU. The R squared value should be greater than 0.98. Include a no cell blank to subtract background luminescence, which should be less than 1 percent of the untreated control signal. Check coefficient of variation (CV) among replicate wells. CV values above 15 percent indicate technical problems such as inconsistent pipetting, edge effects, or cell clumping. If edge effects occur, pre incubate the plate at room temperature or use a plate lid to reduce evaporation during incubation. The Galaxy Training Network provides resources for statistical quality control of high throughput data that can be adapted to viability assays.

Common Mistakes

  • Bubbles in wells: Bubbles scatter light and cause erroneous low readings. Tap the plate gently after adding reagent or use a brief centrifugation at 300 × g for 1 minute.
  • Inadequate mixing: Failure to lyse all cells leads to under reporting of viability. Always shake for the recommended time and speed.
  • Reagent degradation: CellTiter Glo is light sensitive and loses activity after repeated freeze thaw cycles. Aliquot reagent upon arrival and store at 20°C. Avoid exposing the reconstituted reagent to prolonged light.
  • Over incubation: The signal decays slowly after 30 minutes. To maintain consistency, read all experimental plates within the same time window after reagent addition.
  • Using wrong plate type: Clear plates allow light transmission between wells and reduce signal. Always use white, opaque walled plates.
  • Ignoring metabolic interference: Some compounds, especially those that affect ATP synthesis (e.g., oligomycin, 2 deoxyglucose), alter ATP levels independently of cell number. Validate results with an orthogonal assay when testing such agents. Research on Combination of Enzastaurin and Ibrutinib synergistically induces anti tumor effects in diffuse large B cell lymphoma used ATP based assays alongside other methods to confirm synergistic cytotoxicity.

Limits of Interpretation

The CellTiter Glo assay measures total ATP from viable cells, but it cannot distinguish between proliferating cells and quiescent cells that maintain ATP. It also does not differentiate between apoptosis and necrosis, as both processes eventually lead to ATP depletion. For kinetic studies, consider that ATP levels may drop during the early phase of apoptosis before cells fully lose membrane integrity. Additionally, the luminescence signal is influenced by cell size: larger cells contain more ATP per cell. Therefore, compare RLU values only between samples of the same cell type under similar conditions. For studies involving cell cycle arrest or differentiation, normalize to cell number determined by a method such as crystal violet staining or nuclear counting. The protocol is less reliable for long term experiments exceeding 5 days because ATP per cell can change due to metabolic adaptation. The Alofanib an allosteric FGFR2 inhibitor has potent effects on ovarian cancer growth in preclinical studies describes a preclinical evaluation that used this assay to measure antiproliferative effects, but the authors also performed colony formation assays to confirm results. Always interpret CellTiter Glo data alongside additional endpoints such as cell cycle analysis, caspase activation, or direct cell counting.

Frequently Asked Questions

Q1. Can I use CellTiter Glo with adherent cells that detach during treatment?
Yes. The assay lyses all cells in the well, including detached cells. However, if many cells detach, the signal may reflect cells in suspension as well as those still attached. Centrifuge the plate briefly (300 × g for 5 minutes) before adding reagent to pellet detached cells at the bottom of the well.

Q2. How do I calculate percent viability?
Subtract the blank RLU (medium only) from all sample RLU values. Then divide the corrected RLU of each treated sample by the corrected RLU of the untreated control and multiply by 100. For dose response curves, fit the data to a sigmoidal model to obtain IC50 values.

Q3. Why is my luminescence signal too high or too low?
Too high: cell density may be too high, leading to ATP saturation of the luciferase reaction. Reduce seeding density or dilute the sample with medium before adding reagent. Too low: insufficient cells, poor cell viability, or reagent degradation. Check cell health before seeding.

Q4. Can I use the protocol for 3D spheroid cultures?
The standard protocol is optimized for monolayer or suspension cultures. For spheroids, dissociation into single cells before the assay is recommended, or use a lysis step that penetrates the spheroid. Some manufacturers provide a 3D version of the reagent. Validate the linearity with spheroids of known cell numbers.

References and Further Reading

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