1. Purpose
This SOP describes the procedure for assessing cell viability and proliferation using the Alamar Blue assay. The method is based on the reduction of resazurin (non-fluorescent, blue) to resorufin (fluorescent, pink) by metabolically active cells. The generated signal is proportional to the number of viable cells.
2. Principle
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Resazurin enters living cells and is reduced by mitochondrial and cytosolic enzymes to resorufin.
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Resorufin can be quantified by measuring fluorescence (Ex 560 nm/Em 590 nm) or absorbance (570 nm and 600 nm).
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Non-viable cells lack reducing capacity and do not convert resazurin.
3. Materials and Equipment
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Cells: e.g., MDA-MB-468 or other mammalian cells
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Complete growth medium (appropriate for cell line)
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Alamar Blue reagent (10× stock, e.g., Invitrogen, Bio-Rad)
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96-well tissue culture plates
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Incubator (37 °C, humidified, 5% CO₂ or per cell line requirements)
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Plate reader (e.g., BioTek Synergy H1) capable of fluorescence or absorbance detection
4. Procedure
4.1 Cell Seeding
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Harvest and count cells.
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Seed 5,000–10,000 cells per well in a 96-well plate in 100 µL of complete medium.
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Include blank wells containing medium only (no cells).
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Incubate cells overnight (12–24 h) to allow attachment (for adherent lines).
4.2 Treatment
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Apply drug as required.
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Maintain appropriate untreated and vehicle controls.
4.3 Alamar Blue Assay
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Add 10 µL of Alamar Blue reagent directly to each well (10% of culture volume).
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Gently tap or shake plate to mix.
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Incubate at 37 °C for 1–4 h (optimal time depends on cell density and metabolic activity).
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Wells should change from blue (oxidized) toward purple/pink (reduced).
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Read signal:
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Fluorescence: Excitation 560 nm, Emission 590 nm (preferred).
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Absorbance: Measure at 570 nm and 600 nm, then calculate reduction percentage using manufacturer’s formula.
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5. Data Analysis
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Subtract background (blank wells without cells).
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Express viability relative to control (e.g., untreated = 100%).
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For drug testing, plot cell viability (%) vs. drug concentration to calculate IC₅₀.
6. Notes and Precautions
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Do not expose Alamar Blue to light for extended periods (light sensitive).
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Avoid bubbles in wells (interfere with readings).
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The assay is non-destructive: plates can be returned to the incubator for repeated measurements.
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Incubation time should be optimized—over-incubation may saturate the signal.
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Perform each condition in triplicate for reliability.