Cell viability assays, glucose uptake and calcium influx assays MCQs With Answer

Introduction

This collection of multiple-choice questions focuses on cell viability assays, glucose uptake methods, and calcium influx measurements—core techniques in Cellular and Molecular Pharmacology for M.Pharm students. The questions probe assay principles, typical reagents, instrumentation, data interpretation, potential artifacts, and appropriate controls. You will find scenario-based items that require deeper understanding of how assay chemistry reflects biological states (e.g., mitochondrial activity vs membrane integrity), how to distinguish transporter-mediated glucose uptake, and how to select and interpret calcium indicators and kinetic measurements. These MCQs are designed to prepare you for exams and practical lab decision-making by emphasizing critical nuances and troubleshooting strategies.

Q1. Which statement best distinguishes MTT assay readout from trypan blue exclusion when assessing cell viability?

• MTT measures membrane integrity while trypan blue measures mitochondrial reductase activity
• MTT measures metabolic reduction by cellular dehydrogenases while trypan blue measures membrane integrity by dye exclusion
• Both MTT and trypan blue directly measure cellular ATP content
• MTT is a direct measure of apoptosis whereas trypan blue quantifies necrosis only

Correct Answer: MTT measures metabolic reduction by cellular dehydrogenases while trypan blue measures membrane integrity by dye exclusion

Q2. In an MTT assay, incomplete solubilization of formazan crystals most likely leads to which artifact?

• Apparent increase in cell viability due to overestimation of absorbance
• Apparent decrease in cell viability due to underestimation of absorbance
• No effect because formazan is water soluble
• Shift in wavelength of absorbance without affecting absorbance intensity

Correct Answer: Apparent decrease in cell viability due to underestimation of absorbance

Q3. Which assay is least suitable if you need a non-destructive, real-time measurement of cell metabolic activity over time?

• Resazurin (Alamar Blue) assay
• Real-time impedance-based cell index assay
• ATP luminescence assay (CellTiter-Glo)
• Fluorescent probe for mitochondrial membrane potential measured in live wells

Correct Answer: ATP luminescence assay (CellTiter-Glo)

Q4. Which of the following is a common interference in colorimetric tetrazolium assays (MTT/XTT/WST) that can produce false-positive signals?

• Presence of phenol red in medium interacting with tetrazolium chemistry
• High extracellular ATP concentration
• Non-metabolized glucose in medium
• Use of EDTA in the reagent formulation

Correct Answer: Presence of phenol red in medium interacting with tetrazolium chemistry

Q5. For measuring glucose uptake via 2-deoxyglucose (2-DG) uptake assay, which control specifically tests transporter-mediated uptake?

• Using cytochalasin B to inhibit GLUT-mediated transport
• Adding excess ATP to the medium
• Treating cells with ionomycin to increase intracellular calcium
• Depleting extracellular sodium with choline chloride

Correct Answer: Using cytochalasin B to inhibit GLUT-mediated transport

Q6. Which labeled glucose analog is fluorescent and commonly used for microscopy-based glucose uptake assays?

• 14C-glucose
• 2-deoxy-D-[3H]glucose
• 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose)
• FDG-PET tracer (18F-FDG)

Correct Answer: 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose)

Q7. When using radiolabeled 2-deoxyglucose (2-[3H]DG) to quantify uptake, which step is essential to distinguish between bound and internalized tracer?

• Lysing cells directly without washes
• Performing an ice-cold wash with phloretin to remove surface-bound tracer
• Heating cells to 95°C to deactivate transporters
• Adding excess non-labeled glucose at the end to outcompete bound radiotracer

Correct Answer: Performing an ice-cold wash with phloretin to remove surface-bound tracer

Q8. Which glucose transporter isoform is primarily responsible for insulin-stimulated glucose uptake in adipose and muscle cells?

• GLUT1
• GLUT2
• GLUT3
• GLUT4

Correct Answer: GLUT4

Q9. What is the main advantage of ratiometric calcium indicators like Fura-2 over single-wavelength dyes like Fluo-4?

• Ratiometric dyes do not require excitation light
• Ratiometric dyes allow correction for dye loading, photobleaching, and variable pathlength by measuring two wavelengths
• Ratiometric dyes are less sensitive to calcium concentration changes
• Ratiometric dyes are non-fluorescent until bound to calcium

Correct Answer: Ratiometric dyes allow correction for dye loading, photobleaching, and variable pathlength by measuring two wavelengths

Q10. Which compound is commonly used as an intracellular calcium chelator to test the dependence of a response on intracellular Ca2+?

• EGTA (extracellular calcium chelator)
• BAPTA-AM (cell-permeant intracellular calcium chelator)
• Lanthanum chloride (extracellular channel blocker)
• Thapsigargin (ER Ca2+ ATPase inhibitor)

Correct Answer: BAPTA-AM (cell-permeant intracellular calcium chelator)

Q11. In a Fluo-4 calcium influx assay on a plate reader, which approach best distinguishes store-operated calcium entry (SOCE) from voltage-gated calcium entry?

• Stimulation with high extracellular potassium to depolarize the membrane
• Depleting ER calcium stores with thapsigargin in Ca2+-free buffer, then reintroducing extracellular Ca2+
• Treating cells with ionomycin in the presence of high extracellular Na+
• Applying insulin to the cells and measuring immediate calcium rise

Correct Answer: Depleting ER calcium stores with thapsigargin in Ca2+-free buffer, then reintroducing extracellular Ca2+

Q12. Which of the following is a pitfall when using ATP-based luminescence assays to estimate viable cell number?

• ATP assay signal is unaffected by metabolic state or cell cycle phase
• Residual ATP from dead cells or extracellular ATPases can alter the signal
• ATP assays are independent of reagent luciferase activity
• ATP luminescence has linearity at extremely high cell densities without saturation

Correct Answer: Residual ATP from dead cells or extracellular ATPases can alter the signal

Q13. When designing a glucose uptake experiment using 2-NBDG, what condition is critical to include to validate specificity of uptake through facilitative glucose transporters?

• Co-incubation with an inhibitor like cytochalasin B or phloretin
• Using serum-containing medium without washing
• Measuring fluorescence only after 24 hours
• Adding excess ATP to the wells during uptake

Correct Answer: Co-incubation with an inhibitor like cytochalasin B or phloretin

Q14. In calcium imaging using Indo-1, what unique detection method is used compared to single-emission dyes?

• Indo-1 is imaged using electron microscopy
• Indo-1 produces two emission peaks allowing ratiometric measurement by measuring emission shift upon Ca2+ binding
• Indo-1 uses chemiluminescence instead of fluorescence
• Indo-1 signals are detected by absorbance changes at 260 nm

Correct Answer: Indo-1 produces two emission peaks allowing ratiometric measurement by measuring emission shift upon Ca2+ binding

Q15. Which parameter is most important when converting raw absorbance from an MTT assay into percent cell viability?

• Subtracting background (blank) absorbance and normalizing to untreated control absorbance
• Dividing absorbance by well volume only
• Using only the highest absorbance values across plate replicates
• Normalizing absorbance to fluorescence units

Correct Answer: Subtracting background (blank) absorbance and normalizing to untreated control absorbance

Q16. Which reagent is typically used to solubilize formazan crystals in an MTT assay for microplate reading?

• Phosphate-buffered saline (PBS) alone
• DMSO or acidified isopropanol
• Pure water at room temperature
• Trypsin-EDTA solution

Correct Answer: DMSO or acidified isopropanol

Q17. In a glucose uptake experiment, why is it important to serum-starve cells prior to insulin-stimulated uptake assays?

• Serum starvation prevents cell adhesion
• Serum starvation depletes intracellular glycogen stores
• Serum starvation reduces basal insulin signaling and GLUT4 translocation, enhancing fold-response to insulin
• Serum starvation increases extracellular glucose concentration

Correct Answer: Serum starvation reduces basal insulin signaling and GLUT4 translocation, enhancing fold-response to insulin

Q18. Which experimental approach helps prevent photobleaching artifacts during live-cell calcium imaging experiments?

• Maximizing laser/excitation intensity to reduce exposure time
• Using the highest dye concentration possible regardless of buffering
• Minimizing excitation intensity and exposure time while using neutral density filters and interval imaging
• Recording continuously at maximal frame rate for long periods

Correct Answer: Minimizing excitation intensity and exposure time while using neutral density filters and interval imaging

Q19. What is a primary reason to include sodium azide or oligomycin controls when interpreting tetrazolium reduction assays?

• They stimulate mitochondrial dehydrogenases to boost signal
• They inhibit mitochondrial respiration thereby demonstrating dependence of tetrazolium reduction on mitochondrial function
• They chelate extracellular calcium to reduce background
• They enhance membrane permeability to increase dye uptake

Correct Answer: They inhibit mitochondrial respiration thereby demonstrating dependence of tetrazolium reduction on mitochondrial function

Q20. When measuring calcium influx with a plate reader using Fluo-4, which calibration step can convert fluorescence units to approximate intracellular [Ca2+]?

• Using only a single-point blank without further reagents
• Applying a two-point calibration with a calcium chelator (EGTA) for Fmin and ionomycin plus excess Ca2+ for Fmax
• Normalizing fluorescence to total protein without dye calibration
• Measuring fluorescence at room temperature and assuming linearity across cell types

Correct Answer: Applying a two-point calibration with a calcium chelator (EGTA) for Fmin and ionomycin plus excess Ca2+ for Fmax

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