Cell Counter

At the Cell Counter: HUVEC Cells

Human umbilical vein endothelial cells (HUVEC) are primary cells isolated from the vein of the umbilical cord. They are a model system for studying endothelial cell function, with applications including hypoxia, inflammation, oxidative stress, response to infection, and both normal and tumor-associated angiogenesis. Endothelial cell activation, an inflammatory response, can be induced by cytokines such as TNF-α and IFN-γ and results in upregulation of cell adhesion molecules like VCAM-1/CD106, whose upregulation can be quantified using fluorescently labeled antibodies and cellular imaging.

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Figure 1: StainFree cell counts

HUVEC were imaged using the SpectraMax i3 MiniMax 300 Imaging Cytometer, and cells were identified from transmitted light images using the predefined ‘CellsB’ analysis setting. Shown on the left are the original transmitted light images, and on the right are the same images with purple masks indicating cells identified by the software. Cells seeded at high (top row) and low (bottom row) densities are shown.

Figure 2: StainFree cell counts vs. fluorescent nuclear counts

HUVEC seeded at densities ranging from 156 to 20,000 cells per well were counted using StainFree technology (blue circles), or they were stained with EarlyTox™ Live Red Dye and red-fluorescent nuclei were counted (red circles). Cell counts obtained with both methods agreed closely across the entire range of cell densities.

Figure 3: Cytokine-induced VCAM-1/CD106 expression in HUVEC

HUVEC treated with cytokines TNF-α and IFN-γ (left) or untreated (right) were stained with FITC-labeled antibody against VCAM-1/CD106 and imaged with the SpectraMax MiniMax 300 Imaging Cytometer.

Figure 4: Inhibition of cytokine-induced VCAM-1/CD106 expression in HUVEC

HUVEC were seeded at 10,000 cells per well in a 96-well plate and allowed to attach and grow overnight. They were treated with the cytokines TNF-α and IFN-γ, as well as different concentrations of the p38 MAP kinase inhibitor SB202190, for 24 hours and then stained with a FITC-labeled antibody to VCAM-1/CD106. Controls without inhibitor or cytokines were included.

Tip :

HUVEC cells have a typical cobblestone appearance. For StainFree counting, the preconfigured setting ‘CellsB’ in the SoftMax Pro Software image analysis settings works very well. Alternatively, when counting cells whose morphology has changed due to treatment with cytokines, etc., creating a new analysis setting by drawing on the cell images may be helpful.

HUVEC Cells Analysis Toolkit

  • SpectraMax® i3 Multi-Mode Microplate Detection Platform
  • SpectraMax® MiniMax™ 300 Imaging Cytometer
  • SoftMax® Pro Software
Parameter Setting for cell counts
Optical configuration SpectraMax MiniMax 300 Imaging Cytometer
Read mode Imaging
Read type Endpoint
Wavelength settings Transmitted light
Image acquisition settings Exposure: 8 ms
Image analysis settings Analysis type: Discrete Object Analysis
Wavelength for finding objects: TL
Find objects Predefined setting: 'CellsB' or drawing

About StainFree Cell Detection Technology

Imaging cell-based assays typically requires the use of fluorescent probes that can be toxic to living cells or may only function in fixed cells. A label-free method for analyzing cell counts and cell confluence enables researchers to quantitatively monitor cell proliferation and health without time-consuming workflows that may disrupt cell viability.

The SpectraMax i3 Multi-Mode Microplate Platform with MiniMax 300 Imaging Cytometer uses unique, patent-pending StainFree Cell Detection Technology that allows you to perform cell proliferation, cytotoxicity, and other assays without nuclear stains like DAPI, which intercalates with DNA, or live cell dyes that are actually toxic to cells in the long term.