Lec 4c LDH and Cell Viability Assays Study Notes

Overview of LDH and Cell Viability Assays

• Importance of LDH in understanding cell viability

  • LDH (lactate dehydrogenase) typically released by dead cells into the surrounding media

  • Assay method involves treatment of cells with compounds (drugs) at various concentrations and time points

Assay Procedure

• Treatment of cells with compounds

  • Transfer supernatant to a new plate after treatment

  • Supernatant contains released LDH (RLTH)

• Addition of reaction mixture from assay kit

  • Incubate for 30 minutes

  • Reaction is stopped and absorbance is measured

Measurement Analysis

• Focus on absorbance measurement to determine cell viability

  • Typical setup for cell viability assay versus LDH assay

• Graphical representation in research papers

  • X-axis: concentration range

  • Y-axis: cell viability

  • Observation: No significant cytotoxic event observed up to 100 μM concentration

  • All concentrations maintain around 100% cell viability (90-100% viable)

• LDH measurement indicating cell death

  • Initial release of LDH shows an increase from 0% to 20% at the lowest concentration

  • Indicates presence of dead cells but majority remain viable

  • Suggests handling effects may initially increase LDH release

• Importance of conducting both ATP assay and LDH assay

  • MTT assay recommended for ease and cost efficiency

Understanding Apoptosis and Necrosis

• Role of mitochondria in apoptosis

  • Mitochondria release cytochrome c leading to controlled programmed cell death (apoptosis)

• Distinction between apoptotic and necrotic zones in drug testing

  • Concentration curve analysis

  • Above 50% viability: apoptosis likely occurring

  • Below 50% viability: cells may enter necrotic phase due to increased drug concentration

• Timing observations with and without drug application

  • Baseline observations show viable cells potentially dip at certain time intervals (24h, 48h, 72h)

  • Changes could be attributed to nutrient depletion or space limitations in culture conditions

Standard Practices in Cell Culture Testing

• Outline of essential tests in cell culture for drug assessments

  • Typical standard tests include ATP, MTT, and LDH assays

  • Overview of each assay type discussed in context of laboratory work with instructor and peers

Calculation Examples for Drug Preparation

Standard Concentration Calculation

• Drug concentration details

  • Initial concentration: 100 mM in DMSO

  • Molecular weight: 386 g/mol

  • Preparation of 5 mL of 100 mM solution

  • Calculate moles needed:

    • Moles = concentration (mol/L) × volume (L)

    • Moles = 0.1 mol/L × 0.005 L = 0.0005 moles

  • Mass = moles × molecular weight

    • Mass = 0.0005 moles × 386 g/mol = 0.193 g of drug needed

  • Dissolve in 5 mL DMSO

Stock to Working Solution Preparation

• Creating a 20 mM working solution from 100 mM stock

  • Required final volume = 10 mL

  • Using formula: C1V1 = C2V2

  • C1 = 100 mM

  • C2 = 20 mM

  • V2 = 10 mL

  • Rearranging affords: V1 = (C2 × V2) / C1

  • V1 = (20 mM × 10 mL) / 100 mM = 2 mL (of 100 mM stock solution)

  • Final volume adjusted with DMSO and media to maintain dilution

Managing DMSO Concentration

• DMSO dilute considerations

  • Initial 100% DMSO concentration lowered to 20% by adding media (8 mL) to 2 mL of stock solution

  • Maintain maximum cell exposure to 1% DMSO during tests

Additional Dilutions for Cytotoxicity Testing

• Preparing dilutions for testing different concentrations

  • Aim for final concentrations per well: 100 μM, 500 μM, 150 μM

• Calculate volumes needed for each final concentration using dilution equations

Conclusion and Study Recommendations

• Importance of understanding drug effects on cell viability

• Familiarity with calculation methods for drug preparation and DMSO management

• Emphasis on laboratory practice and testing of drug concentrations on cell cultures in upcoming labs with instructor