Cell Proliferation Assay Services

DNA Synthesis Detection

BrdU incorporation assay:

• Principle: Bromodeoxyuridine (BrdU), a thymidine analog, is incorporated into newly synthesized DNA strands during the S phase. Immunofluorescence and flow cytometry enable detection of BrdU-labeled cells which measure proliferation activity.
• Procedure: Cell culture → BrdU pulse labeling → Fixation/permeabilization → Anti-BrdU antibody incubation → Fluorescence detection/flow analysis.

Fig. 1. The BrdU incorporation process (Yu JH, Wang ZX, et al., 2022).

Cell Metabolism Detection

XTT and MTT assay:

• Principle: XTT (tetrazolium salt) and MTT (thiazolyl blue) are reduced by mitochondrial dehydrogenases in live cells to soluble or insoluble formazan products, with intensity correlating to cell number.
• Procedure: Cell culture → XTT/MTT reagent addition → Incubation → For MTT, dissolve formazan → Spectrophotometer or plate reader detection (XTT: 450 nm, MTT: 570 nm).

Resazurin assay:

• Principle: Resazurin, a blue precursor, is metabolized by live cells into highly fluorescent resorufin, correlating with metabolic activity.
• Procedure: Cell culture → Resazurin reagent addition → Incubation → Fluorescence detection (Excitation: 530 nm, Emission: 590 nm).

Fig. 2. The principle of XTT and resazurin assay (Jaśkiewicz M, Janczura A, et al., 2019).

Alamar blue assay:

• Principle: Alamar Blue functions as a cell viability fluorescent indicator by undergoing reduction through mitochondrial dehydrogenases to form a red fluorescent compound which demonstrates a direct relationship with both cell numbers and metabolic rates.
• Procedure: Cell culture → Alamar Blue addition → Incubation → Fluorometer or fluorescent plate reader detection (Excitation: 530 nm, Emission: 590 nm).

ATP Concentration Detection:

ATP luminescence assay:

• Principle: ATP functions as an essential component of cellular energy metabolism while its levels show a direct relationship with both cellular activity and cell numbers. ATP activates luciferase which oxidizes luciferin to produce luminescence whose intensity reveals ATP levels and thus estimates live cell numbers.
• Procedure: Cell lysis → Luciferase reagent addition → Incubation → Luminescence detection (chemiluminescence instrument).

Fig. 3. Schematic illustration of the principles of ATP assay (Kamiloglu S, Sari G, et al., 2020).

Cell Tracing Detection:

CFSE assay:

• Principle: Carboxyfluorescein diacetate succinimidyl ester (CFSE) enters cells and hydrolyzes into fluorescent molecules, distributing equally during division and decreasing in intensity to reflect generational proliferation.
• Procedure: Cell labeling with CFSE → Culturing → Flow cytometry detection of fluorescence decay.

Fig. 4. Lymphocyte proliferation assay—CFSE assay (Ganesan N, Ronsmans S, et al., 2023)

Cell Membrane Integrity Detection:

LDH release assay:

• Principle: Lactate dehydrogenase (LDH), a cytosolic enzyme, remains within cells when membranes are intact. Membrane damage releases LDH into culture supernatant. LDH activity in supernatant reflects membrane damage extent.
• Procedure: Cell culture → Supernatant collection → LDH substrate addition → Measure lactate dehydrogenase activity → Calculate membrane damage extent.

Fig. 5. Schematic representation of the principle of the LDH release assay (Forest V, Figarol A, et al., 2015).

Trypan blue staining assay:

• Principle: Viable cell membranes block Trypan Blue, while nonviable or damaged cells allow dye entry, staining cells blue.
• Procedure: Mix cell suspension with Trypan Blue dye → Viable cells remain unstained, nonviable cells stain → Microscope observation and count.

Proliferation Marker Detection:

• Principle: Proliferation markers like Ki-67, PCNA, and Cyclin D1 are key proteins in cell cycle regulation, with expression levels closely tied to proliferation status. Examining these markers' expression elucidates cell cycle regulation and evaluates drug effects.
• Procedures:
  Immunofluorescence: Cell culture → Fixation/permeabilization → Primary antibody (Ki-67/PCNA/Cyclin D1) incubation → Fluorescent secondary antibody incubation → Fluorescence microscopy imaging.
  Western Blot: Cell lysis → Protein extraction → SDS-PAGE electrophoresis → Transfer → Primary/secondary antibody incubation → Chemiluminescence detection.
  Flow Cytometry: Cell culture → Fixation/permeabilization → Intracellular antigen labeling antibody incubation → Flow analysis.

3D Cell Proliferation Assays:

Creative Bioarray also offers 3D cell proliferation assays using the Soft Agar assay with optimized growth and assay conditions based on our 3D culture experience. A 2D vs 3D comparison can also be included.