Cell Proliferation and Toxicity Detection Techniques: A Comprehensive Guide to CCK-8 Experimental Procedures

Overview of Experimental Methods and Background Introduction

In modern cell biology research, accurately assessing cell proliferation activity and compound cytotoxicity is a fundamental and critical experimental step. The mainstream detection methods currently include two major systems: MTT method and CCK-8 method. Compared with the former, the CCK-8 method has become a routine choice in laboratories due to its unique advantages. This method was first developed by the Japanese Institute of Chemical Research, based on the color development principle of water-soluble tetrazolium salt WST-8, which quantifies cell status by detecting mitochondrial dehydrogenase activity in viable cells.

The core advantages of the CCK-8 method are reflected in three aspects: firstly, safety; throughout the experiment process there is no need for organic solvents like isopropanol or DMSO used in MTT that dissolve formazan crystals, thus avoiding toxic reagent exposure risks; secondly, sensitivity; it can detect as low as 100 cells/well, making it particularly suitable for low-density cell culture assays; lastly, convenience; reagents can be directly added to culture media for color development without requiring crystallization dissolution steps involved in MTT methodology—greatly simplifying operational procedures. These characteristics have led to its widespread application in drug screening, toxicity assessment, tumor research among other fields.

In-depth Analysis of Experimental Principles

The key component of the CCK-8 kit is water-soluble tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonatophenyl)-2H-tetrazole monosodium salt), which serves as a novel electron coupling reagent. When viable cells are present, succinate dehydrogenase within mitochondria reduces WST-8 into highly soluble orange-yellow formazan products. The extent of this reduction reaction correlates positively with live cell numbers while inversely correlating with levels of cytotoxicity.

It’s noteworthy that this color development reaction requires an electron mediator 1-Methoxy PMS (1-Methoxy phenazine methosulfate) to proceed efficiently. This electron transfer system enhances sensitivity compared to traditional MTT methods by 2–4 times while ensuring uniform distribution of colored products within culture media—avoiding uneven precipitation issues caused by MTT's crystalline form. After coloration occurs, absorbance can be measured at 450nm using an enzyme-labeled instrument allowing accurate data acquisition through standard curves.

Detailed Operational Procedure

Standardized Operation for Cell Suspension Preparation The initial stage involves preparing a uniform single-cell suspension. For suspension cell lines select logarithmic phase cells after centrifuging at 800–1000rpm for five minutes collecting pellets; adherent cells require digestion using 0.25% trypsin (digestion time adjusted according to cell type typically between one-three minutes), followed by adding serum-containing medium halting digestion before similarly centrifuging collection processes ensue again ensuring accurate density measurements via hemocytometer or automated counters adjusting final concentrations ranging from (5×10^3)~(5×10^4)cells/ml guaranteeing optimal conditions achieving approximately seventy-eighty percent confluence post twenty-four hours incubation period on ninety-six well plates ideal state detected during experiments. Cell Seeding & Group Design Using sterile ninety-six well plates during experimentation each well should receive one hundred microliters’ worth cellular suspensions applied carefully preventing bubble formation disrupting subsequent analyses via pipette tips contacting sidewalls slowly dispensing samples instead maintaining appropriate controls including blank control group (media only), negative control group (cells + media), positive control group(cells + known toxic compounds) alongside test drug groups ideally replicating six wells per condition enhancing reliability results derived from testing protocols following log dilution principles evaluating dose-response relationships thoroughly across five-seven concentration gradients such as (100μM,) (10μM,) (1μM). ... [Content continues] ...