This protocol provides the required information to obtain a single cell analysis profile for HUVEC which have been exposed to differential levels of induced inflammation via lipopolysaccharide (LPS) exposure.
Method Article
Human Umbilical Vein Endothelial Cells (HUVEC) Label Free Single Cell Photonic Fingerprinting
https://doi.org/10.1038/protex.2015.123
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posted 22 Dec, 2015
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This protocol provides the required information to obtain a single cell analysis profile for HUVEC which have been exposed to differential levels of induced inflammation via lipopolysaccharide (LPS) exposure.
autofluorescence
lipopolysaccharide
inflammation
broadband light interrogation
cell array
Reagent preparation protocol
Differential inflammation has been implemented by exposing the cells to various concentrations of LPS during the cell culture process (below). LPS was obtained from Sigma Aldrich Ireland (L2654-1MG) and was diluted to a final stock concentration of 1,000 μg/mL. A further dilution of stock concentration to diluent at a ratio of 1:5 was then applied to give a working concentration of 200 µg/mL. The diluent used was distilled water (DH2O) with 0.1% BSA, twice filtered with a 0.22 μm filter. Each concentration of LPS added to the cells under culture was incubated for a 24-hour period under the conditions described below.
The fluidic volumes of LPS, along with their corresponding concentrations for a 20-mL cell culture flask are summarised in Table 1.
See figure in Figures section.Cell culture protocol
HUVEC cells were obtained from PromoCell GmbH, Germany (C-12203 HUVEC-c pooled). Cells were cultured in PromoCell Endothelial Cell Growth Medium (C22110), supplemented with Fetal Calf Serum (0.02mL/mL), Endothelial Cell Growth Supplement (0.004 mL/mL), Epidermal Growth Factor (recombinant human) (0.1ng/mL), Basic Fibroblast Growth Factor (recombinant human) (1ng/mL), Heparin (90µg/mL), and Hydrocortisone (1µg/mL). Cells were cultured in 75cm2 flasks at 37°C and 5% CO2. When harvesting or passaging the HUVEC cells, the PromoCell Detach Kit (C41200) was used. Cells were isolated by centrifugation at 220 x g for 3 minutes. The cell pellet was finally resuspended in culture media.
Biochip preparation
Full details of the V-SCA-EC BDT have been provided in D3.2.
Biochip Microfluidic Testing
Table 2: Centrifugal Test Stand Testing Protocol
See figure in Figures section.Figure 1: LiPhos single cell viewgraph. (a) Biochip schematic. (b) Three biochips under centrifugal microfluidic testing. (c) Single cell capture, analysis and PIN readout principles.
See figure in Figures section.Figure 2: V-cup array occupancy distribution for 20-µm polystyrene beads, HUVEC and EA.hy926 cells. Each test case shows an occupancy rate (one bead / cell per cup) of >95%. An empty V-cup or V-cup containing more than one bead / cell is classified as an error. N = 10, p < 0.05.
Optical Measurement Protocol
System measurements
The optical detection system utilised has been described in D3.2 and is summarised in Figure 3. The operational wavelengths utilised are summarised in Table 3. The camera settings for each measurement are summarised in Table 4.
See figure in Figures section.Figure 3: (a) Setup of the centrifugal test stand with integrated fluorescence detection and optical tweezers module. (b) Detailed view of the optical module for fluorescent imaging and particle manipulation. (c) Schematic of the optical system. (c) Royal Society of Chemistry 2015 [1] Burger R., Kurzbuch D., Gorkin R., Kijanka G., Glynn M., Mc Donagh C. and Ducrée J., An Integrated Centrifugo-opto-microfluidic Platform for Arraying, Analysis, Identification and Manipulation of Individual Cells, Lab On A Chip 2015, 15, 378-381.
See figure in Figures section.Table 3: Operational wavelengths utilised for single cell analysis
See figure in Figures section.Table 4: Excitation and detection parameters utilised for single cell analysis
Image Analysis
Images were analysed by the software ImageJ (version 1.46r). Cell signal intensity was quantitated by ‘region of interest (ROI)’ analysis. Background signal levels are removed and an average intensity was obtained for each cell through the time series.
Data Presentation
Bright field imaging (BFI) records cell morphology which changes with the level of cell inflammation which, in turn, leads to an increased number of mitochondria (Fig. 4). Whilst changes are present and detected, they are not as pronounced as the corresponding changes observed under TNF-α exposure. Broadband light interrogation (BBLI) measures the scattering and absorbance bands (Fig. 5) and it is observed that light transmission is lowest for healthy HUVEC and increases with the inflammation-induced protein levels in the cells. AF intensities grow with the concentrations of LPS in each of the three wavelength bands of interest (Fig. 6) but the AF signal magnitudes are smaller than those observed under TNF-α inflammation conditions.
See figure in Figures section.Figure 4: HUVEC which are (a) healthy and treated with LPS for 24 hours at (b) 5 ng/mL and (c) 10 ng/mL. Cell morphology changes are present and detected for increasing levels of LPS exposure; the observed changes are not as pronounced as the changes observed under TNF-α exposure.
See figure in Figures section.Figure 5: Single cell profiles for broadband light (λex = 360 – 800nm) interrogation measuring inherent absorbance and scattering bands for HUVECs treated for various concentrations of LPS. Light transmission is lowest for healthy HUVECs and increases with the inflammation induced protein levels in the cells. (For each cell test condition: n = 210 cells, p<0.05)
See figure in Figures section.Figure 6: Single cell auto-fluorescence profiles for HUVEC treated for various concentrations of LPS. The presence and levels of induced inflammation are confirmed by an increase in auto-fluorescence on each of the wavelength bands examined. Each cell test condition produces a unique photonic signal response. (n = 210 cells, p<0.05)
This work has been partly funded by the European Commission (LiPhos project, www.liphos.eu, Contract No. 317916).
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This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 22 Dec, 2015
You are reading this latest protocol version