This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.
We present a method for live image analysis of LDL cholesterol uptake into various human cell types. This allows to screen for compounds, that modify the transport of LDL cholesterol. The main advantage of using live cell imaging for LDL influx quantification, is that it allows monitoring cell morphology throughout the assay duration, so potentially the toxic compounds can be detected.
To begin, aspirate the media off the previously prepared cells, and wash the cells with 5 milliliters of Dulbecco's phosphate buffered saline. Aspirate the DPDS. Next to detach the cells from the 100 millimeter dish, add 1.5 milliliters of 0.25%Trypsin-EDTA for the HepG2 cells, and add 1.5 milliliters of 0.05%Trypsin-EDTA solution for the HK2 cells, or the HCAECs.
Incubate the plates at 37 degrees celsius for four minutes. Then neutralize the Trypsin by adding 3 milliliters of complete media for the HepG2 and HCAEC cells. Use 3 milliliters of the Trypsin neutralizing solution, compromise the DPDS with 5%fetal bovine serum or FBS for the HK2 cells.
Transfer the cells into 15 milliliter conical tubes, then centrifuge the tubes at 250g for five minutes. After the spin, aspirate the media, and resuspend the cell pellet in complete media. Filter the cells suspension gently through a 40 micron mesh strainer to break up cell clumps.
Count the cells and plate them in an optimized density in a 24-well plate. Incubate the plate overnight at 37 degree celsius to allow the cells to attach. The next day, change the cell media to the base media without FBS for the cell line, plus 5%lipoprotein deficient serum, or 2%FBS media.
Use 500 microliters of total media per well in a 24-well plate. Then continue the incubation for 24 hours to starve the cells. Along with the starvation step or the following day, cells may be treated with desired compounds and appropriate controls.
24 hours after starvation, add 5 microliters of pHrodo red labeled LDL to each well containing 500 microliters of media to obtain a final concentration of 10 micrograms per milliliter. Carefully remove any bubbles from the wells. Immediately after adding the labeled LDL, place the plate in the live cell analysis system incubator and allow the plate to equilibrate for 15 minutes to reduce condensation in the plate.
While the cells are incubating, open the software and schedule the scan by adding the vessel holding the plate. Image 16 images per well, add one hour intervals at 10x magnification for four hours using the red and phase channels. Then create a plate map to use for data processing, select the properties tab and choose the plate map.
Input the cell type in the cells tab and treatments in the compound tap. Then click on the regions tab, select each set of replicates, and assign regions. Once an experimental run is complete, create an image set in the software to train the computer to quantify each parameter included in the counting set.
In the software, open the plate view, then in the analysis job utilities box, choose create or add to image collection. Select new image collection, type a name for the image collection, and choose the red and phase channels by checking the boxes next to the channels. Select five more images from different wells and time points and add them to the image collection by adding to the current image collection.
In the analysis job utilities box, choose new processing definition, select the image collection from the drop-down menu, input the parameters for the type of cells. In the preview box, use the drop-down menu to select preview all. In the analysis mask box, check the confluence mask and the red object mask boxes to view the area included in the analysis for the processing definition.
Scroll through the image collection to ensure the cells and LDL are included in the mask. Select file and save the processing definition. Next, open the experimenting plate view to analyze the set of images from the experimental run.
In the analysis job utilities box, choose launch new analysis job, and select the saved processing definition. Name the analysis job, choose the time range for analysis, and highlight the experimental wells to analyze. Select the launch button.
Once the analysis job is complete, export the data, select the completed analysis and press view. In the utilities menu, choose metric graph export. In the regions menu, choose all wells and in the group menu, to obtain the mean value for each set of wells as a group, choose replicates.
And for exporting the individual values for each well, choose none. In the red object metric menu, choose the total red object integrated intensity. Select the data export button.
Check break data down into individual images. The data is automatically copied on a clipboard and can be pasted to a new spreadsheet file. In the phase object metric menu, choose the confluence, then click the data export button.
In the prompt tab, check break data down into individual images. The data is automatically copied on a clipboard and can be pasted to the spreadsheet file containing total red object integrated intensity data. Now the exported data can be normalized, according to steps 4.1.7 and 4.1.8 of the protocol, to obtain a final LDL uptake values.
Following treatment with Dynasore or recombinant PCSK9, all three tested human cell lines showed significant reductions in LDL influx over a four hour time course. The LDL influxes reduced in human hepatic carcinoma or HepG2 cells, with treatment of Dynasore over the time course, compared to the cells treated with DMSO as a control. Live cell imaging of the LDL influx in HepG2 cells at different time points showed a market increase in LDL uptake following treatment with Simvastatin supporting the sensitivity of this method to detect significant alterations in the LDL influx.
Real-time images investigated at the initial time point and final end point, confirm the normal morphology of the cells following Dynasore treatment, indicating the effectiveness and the safety of this compound. After adding further red labeled LDL to the cells to preserve it's fluorescent activity, it's important to protect the plate from light until it is placed in the live cell imaging system incubator. Cells from different organs can respond differently to treatments, meaning in opposite directions, or they may respond in the same directions but show different rates of change in cholesterol uptake.
Only with the live cell imaging can such differences be detected and accurately quantified.
