Cell Viability and Cytotoxicity Assays: Trypan Blue Exclusion & MTT Assay
Accurate determination of cell viability is foundational to cell culture, pharmacology, toxicology, and oncology. Two distinct principles are utilized:
1. Trypan Blue Dye Exclusion (Membrane Integrity): Trypan Blue is a negatively charged, hydrophilic tetrasodium diazo dye (MW 872 Da). Viable living cells with an intact, functional phospholipid bilayer actively exclude the dye and appear clear, refractive, and bright under light microscopy. In contrast, non-viable, necrotic, or dying cells with compromised plasma membrane integrity take up the dye; the chromophore complexes with intracellular proteins, staining non-viable cells dark blue.
Using an Improved Neubauer Hemocytometer, cells are counted in the four 1.0 mm2 corner squares. Because the depth between chamber floor and coverslip is exactly $0.1,text{mm}$, the volume of one large square is $1,text{mm} times 1,text{mm} times 0.1,text{mm} = 0.1,text{mm}^3 = 10^{-4},text{mL}$.
2. MTT Colorimetric Assay (Mitochondrial Metabolism): The MTT assay evaluates the metabolic competency of viable cells. Water-soluble, yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) readily permeates cell membranes. Active mitochondrial dehydrogenases (principally succinate dehydrogenase) cleave the tetrazolium ring via NADH/NADPH reduction, producing water-insoluble, crystalline purple formazan. Formazan crystals are solubilized in dimethyl sulfoxide (DMSO) and quantified spectrophotometrically at 570 nm.
- 0.4% (w/v) Trypan Blue Solution: Filtered 0.4 g Trypan blue in 100 mL phosphate-buffered saline (PBS, pH 7.4).
- MTT Stock Solution (5 mg/mL): 50 mg MTT powder dissolved in 10 mL PBS, sterile-filtered ($0.22,mutext{m}$), stored in foil-wrapped amber tube at 4°C.
- Dimethyl sulfoxide (DMSO, analytical grade).
- Cell culture suspension (Jurkat, HeLa, or baker’s yeast Saccharomyces cerevisiae).
- Improved Neubauer Hemocytometer with optically flat thick coverslip
- Inverted light microscope / standard compound light microscope
- Microplate spectrophotometer (ELISA reader) at 570 nm (reference 630 nm)
- Precision micropipettes ($10,mutext{L}, 100,mutext{L}, 1000,mutext{L}$), 96-well flat-bottom culture plates
- Hemocytometer Setup: Clean the hemocytometer grid and specialized thick coverslip with 70% ethanol and dry with optical paper. Affix the coverslip over the grid chambers until visible rainbow diffraction patterns (Newton’s rings) appear, confirming precise 0.1 mm chamber depth.
- Dye-Cell Mixing (1:1 Dilution): Thoroughly suspend cells by gentle pipetting. In a 0.5 mL microcentrifuge tube, mix $50,mutext{L}$ of cell suspension with $50,mutext{L}$ of 0.4% Trypan Blue solution (dilution factor $= 2$). Incubate at room temperature for 3 minutes (do NOT exceed 5 minutes).
- Chamber Loading: Draw $10,mutext{L}$ of the mixture into a micropipette. Place tip against the V-shaped loading notch of the hemocytometer. Allow fluid to fill the counting grid by capillary action without overflowing into side moats.
- Systematic Microscopic Counting: Focus under 10× objective. Count cells in the 4 large corner squares (each containing 16 smaller squares). Follow the inclusion/exclusion rule: count cells touching the top and left boundary lines; omit cells touching the bottom and right lines. Record unstained (clear/bright) and blue (dead) cells separately.
- MTT Assay Execution: Seed cells ($1 times 10^4$ cells/well) into a 96-well plate in $100,mutext{L}$ medium. Add $10,mutext{L}$ of 5 mg/mL MTT stock solution to each well. Incubate plate at 37°C in 5% $text{CO}_2$ incubator for 3–4 hours. Carefully aspirate medium without disturbing purple formazan crystals. Add $100,mutext{L}$ DMSO to solubilize crystals; shake plate on orbital shaker for 10 minutes. Read optical density at 570 nm.
| Corner Square | Viable (Clear) Cells | Non-Viable (Blue) Cells | Total Cells | Square Viability (%) |
|---|---|---|---|---|
| Square 1 (Top-Left) | 54 | 6 | 60 | 90.0% |
| Square 2 (Top-Right) | 48 | 5 | 53 | 90.6% |
| Square 3 (Bottom-Left) | 51 | 7 | 58 | 87.9% |
| Square 4 (Bottom-Right) | 55 | 4 | 59 | 93.2% |
| Grand Total (Sum) | 208 viable | 22 dead | 230 total | Mean Viability = 90.4% |
- Incubation Time: Trypan Blue is cytotoxic to living cells upon prolonged exposure. If counted beyond 10 minutes, viable cells begin internalizing the dye, causing falsely low viability scores.
- Overfilling Hemocytometer: If the cell suspension floods into the side moats or lifts the coverslip, the chamber volume exceeds $0.1,text{mm}^3$, generating massive concentration calculation errors.
- Cell Clumping: If cells are clumped, vortexing or gentle trituration through a 25G needle is required before loading; do not count aggregates of $> 3$ cells as single events.
