To analyze the cellular mechanisms of internalization of nanoparticles and bacteria by multispectral imaging flow cytometry. Macrophages are first pretreated with cyto klain D to inhibit actin polymerization. Nanoparticles or salmonella are added to the macrophage, monolayer and incubated.
Then the macrophages are harvested, fixed, and labeled for analysis using an image stream. Multispectral imaging flow cytometer cells that have internalized nanoparticles and or salmonella are distinguished from those with surface bound nanoparticles and or salmonella. The resulting data indicate that both salmonella and nanoparticles are internalized only by cells that have not been treated with cyto klain.
D suggesting that internalization is actin dependent. This technique has several advantages over existing methods such as flow cytometry and confocal microscopy. Primarily, it provides an accurate measure of fluorescent signal intensities and spatial resolution between different cellular features at high speed.
This method can help answer key questions for both biomaterials vaccine and drug delivery research, as well as host of pathogen interaction studies. This includes delineating the internalization pathways utilized by poly anhydride nanoparticles, and bacteria. Generally, individuals new to this method often struggle.
It's important to spend time titrating the dyes in order to obtain optimal fluorescence signals. Also, getting to know the software and creating analysis templates takes time. We first had the idea for this method when we found that poly hydro nanoparticles exhibit pathogen mimicking characteristics while employing microscopy and other techniques.
We then wanted a high throughput system to compare the internalization processes used by salmonella and the pollen hydride nanoparticles Prior to performing the assay. All mammalian and bacterial cell cultures should be harvested and nanoparticle suspensions should be prepared. Begin by harvesting confluent RA W2 64.7 cells using a cell scraper.
Determine the cell number using a hemo cytometer. Then plate the cells in a 24 well culture dish at a density of five times 10 to the fifth cells per well in 0.5 milliliters. Complete DMEM incubate overnight at 37 degrees Celsius and a 5%CO2 incubator.
To prepare the salmonella tarica dilute transform salmonella in C-D-M-E-M to obtain a multiplicity of infection of a hundred per RA W2 64.7 cells in a 16 by 125 millimeter autoclave screw cap glass culture tube. Next, using sterilized whey paper, weigh out five milligrams of 1%FITC loaded poly anhydride nanoparticles generated as described by rean colleagues in their 2009 publication and pharmaceutical research in a 1.5 milliliter micro centrifuge tube. Add the nanoparticles to 0.5 milliliters of cold phosphate buffered saline, and place it on ice.
Keeping the tube on ice. Use an ultrasonic liquid processor with a micro tip to sonicate the suspension for approximately 25 seconds at four to six joules. Now that all the needed cells and reagents are ready, the phagocytosis assay can be performed.
Label 24, well tissue culture plates containing cultured RA W2 64.7 cells in preparation for the assay on the first plate. Each of the following samples will be assayed in triplicate, cyto and D with nanoparticles, cyto and D with salmonella, medium with nanoparticles, medium with salmonella nanoparticles only, salmonella only, and AF six 60 stain cells only four degrees Celsius controls will be assayed on the second plate and will include medium plus nanoparticles and medium plus salmonella incubation at this low temperature, slow cellular processes such as phagocytosis one hour prior to induction. Add five micrograms per milliliters of cyto and D and C-D-M-E-M to the appropriate wells and incubate the cells at 37 degrees Celsius.
After the incubation, vortex the nanoparticle suspension and add 10 microliters to the appropriate wells. Then vortex the salmonella and add it to the appropriate wells at an MOI of 100. Tap the plate a few times to mix.
Then place the sample plates at 37 degrees Celsius and the negative control plates at four degrees Celsius for 45 minutes. Following the incubation, place the plates on ice, wash the cells twice with ice cold PBS without calcium and magnesium by aspirating and discarding the old medium to remove unbound particles, salmonella, and dead or detached cells. Use of petal magnesium.
Pre PBS at this stage is essential because it facilitates cell detachment from the substrate To harvest the cells, add 250 microliters of ice cold PBS and gently scrape the wells, pipette the harvested cells into siliconized snap cap micro centrifuge tubes, and keep them on ice. Wash the cells by adding one milliliter of cold wash buffer, then centrifuge at 250 times G for 10 minutes at four degrees Celsius. After discarding the supernatant, remove the residual buffer by tapping the inverted micro centrifuge tube on paper.
Towel resuspend the cell pellet by gently raking the micro centrifuge tube across a test tube rack. To fix the cells, add 100 microliters of 4%para formaldehyde in PBS and allow the cells to stand for 15 minutes. At room temperature, wash the cells by adding one milliliter of perm wash buffer, then centrifuge at 250 times G for 10 minutes at four degrees Celsius.
After discarding the supernatant, remove the residual buffer as before. Then to stain the RA W2 64.7 cells. For actin, add 100 microliters of perm wash buffer containing Alexa Fluora foid in six 60 for 15 minutes.
At room temperature, the unstained samples should be incubated with perm, wash buffer without phin. After staining, wash and centrifuge the cells again, then resuspend them in 50 microliters of PBS containing 1%PFA and store them in the dark at four degrees Celsius until acquisition. Power up, image stream and launch.
Inspire and initialize the fluidics in the file menu. Choose load default template. In the image gallery, view menu, select all and press run setup to start imaging the beads if necessary, adjust the core tracking to center images laterally.
Select the brightfield channel and click set intensity. Wait until the flow speed coefficient of variation is consistently less than 0.2%In the assist tab, click start all to run calibrations and tests and verify that all have passed in the software. Click on load sample.
Then when instructed to do so, place the vial containing the brightest sample. With all of the fluorochromes in the sample loader, select 40 x magnification. Once the instrument settings are established, do not change them for the entire experiment.
Turn on each laser in the experiment by clicking on the laser icons in the software and set the laser power so each fluorochrome has max pixel values between 104, 000 counts as measured in the scatterplots. To eliminate collection of unwanted objects, click on the cell classifier window in the software. Then to collect only cell data, select the area lower limit channel one for brightfield, and set the value to 50 micrometers objects with area less than 50 micrometers will be considered debris and will not be acquired.
Select channels to be collected by clicking the appropriate channel icons in the software here. Channels 1, 2, 3, 4, 5, and six are selected under the setup tab. Enter the file number and destination folder.
Set the sequence number to one and the number of events to acquire to 5, 000. Click on run, acquire to collect and save the first experiment data file. Click FLL and run the next experimental sample.
Repeat until all experimental samples have been collected. Next, to run the controls, click on comp settings. This will turn off the bright field and scatter laser and enable collection of all fluorescence channels under the acquire tab.
Change the input value to 500. Then place control tube and click on run. Acquire to collect 500 positive cells in single stained controls.
Repeat for each fluorochrome in the experiment to develop a compensation matrix. Once all of the sample images have been acquired, launch the ideas analysis software on a separate analysis computer by double clicking on the ideas icon on the desktop. Double click on the internalization wizard and load one of the test sample RIF files.
A popup window will provide instructions for loading the test files. Follow the instructions and click on the next button. Next, click on new matrix.
This will launch the compensation wizard. When prompted, select the data files for the single color controls to add them. Click next through the wizard following directions until the compensation matrix file is saved and loaded into the box in step two of the internalization wizard.
Click next and follow the directions until A DAF file is generated. Set image display properties by selecting the image channels used during acquisition. Click on channel two for FITC and channel five for AF six 60.
Brightfield and side scatter are selected by default. Select the Brightfield channel O one for making the cell boundary and the channel O2 in which nanoparticles or bacteria were collected for the internalizing probe to define the single cell population, a scatter plot of brightfield area versus brightfield aspect ratio of all the cells is generated. The high throughput analysis of multiple data files requires a template file, and so it is of critical importance to carefully define the gates while creating a template file.
Each dot represents the values for an individual cell image. Click on a single. in a graph to select the image of that particular cell in the image gallery.
Next, draw a gate around cells with the area and aspect ratio that corresponds to single cell events. Single cells have an aspect ratio of round one and doublets. Have an aspect ratio of around 0.5.
Click on multiple cells to differentiate between the region containing single cells versus doublets or debris. To generate a histogram of the brightfield gradient root means square of the brightfield image. Click on the next button.
Then under the population tab, select the bin option to display the selected bin. Click on the bins to determine where cells and best focus. Begin and draw a line region to gait focused cells.
The higher the gradient RMS, the better focused, skip the next step unless there are other stains to gate on. A new scatterplot of intensity of channel two on the x axis versus max pixel of channel two on the Y axis is generated. Click on the dots and view the images to help determine the region to draw around the cells that are positive for nanoparticles or bacteria.
A histogram of the internalization feature is generated with a region that begins at zero, which should be adjusted by observing images. The internalization feature is a ratio of the intensity inside the cell to the intensity of the whole cell. It is scaled such that at a value of zero half of the intensity is inside.
The wizard has created a region of each cell that designates the inside by making a mask that is used, the cell image input to find the cell surface and eroded this by four pixels. Note that this mask can be manually adjusted for different cell types when necessary by creating an object mask on the brightfield image first and eroded this by more or fewer pixels. The internalization feature is calculated based on this eroded object mask using an algorithm in the software.
This feature allows us to distinguish internalized particles and bacteria which have the majority of their fluorescent signal within the mask boundary from surface bound particles and bacteria, which have the majority of their fluorescent signal outside the mask boundary as shown in this example, create a new histogram with a new internalization feature based on the eroded object mask. Draw a region to gate on internalized cells by viewing the imagery in selected bin mode. Set the gate at 0.3.Here.
Cells with a score lower than 0.3 are considered surface bound particle positive cells. To eliminate the cells with background labeling and to identify specific internalized nanoparticles or bacteria, choose features from the analysis menu. Click on the analysis menu.
Then click on the spot count feature next to add the mean spot count to the statistics report. In the reports menu, click on the reports icon. Then define statistics report, then add columns, then select on the appropriate cell population.
Click okay. The internalization wizard will automatically add statistics to this report to save the data file as template file to be used for batch analysis of all experimental files. Click on the file menu and select save as template.
Template files. Have the extension dot AST to analyze multiple data files in the idea software. Click tools and select batch data files and input all the RIF files.
Add the compensation matrix file and the template file in the corresponding sections to submit the batch for processing, click submit. After the processing step, all the RAF files are analyzed and DAF files are generated for each of the individual raw files. A final report is generated with statistics for all the samples to compare the effect of actin inhibition and low temperature on the phagocytosis of salmonella and nanoparticles.
RA W2 64.7 cells were incubated at 37 degrees Celsius in medium, either with or without cyto klain D or incubated in medium at four degrees celsius. Both the temperature and the actin manipulations reduced internalization of both nanoparticles and salmonella. However, incubating the cells in cyto and D increase the percent of cells with surface bound nanoparticles while decreasing the percent of cells with surface bound salmonella.
The percentage of cells positive for surface bound nanoparticles increase from approximately 8%at 37 degrees Celsius to greater than 35%after either cyto and D or four degrees Celsius treatment. In contrast, the percentage of cells with surface bound salmonella was reduced from 35%to 15%Following cyto and D treatment, incubation of RA W2 64.7 cells with salmonella at four degrees Celsius, decreased internalization without an apparent increase in the amount of surface bound bacteria as compared to the 37 degrees Celsius control. Together, these data demonstrate that salmonella and nanoparticles are internalized by a similar cellular process that requires actin and is temperature dependent.
Moreover, the data indicate that sustained attachment of salmonella to macrophages requires actin polymerization. After watching this video, you should have a good understanding of how to exa the cellular internalization of nanoparticles and bacteria by multispectral imaging flow cytometry. While attempting this procedure, it is important to remember that inhibitors are cytotoxic.
Therefore, prior cytotoxicity profiling experiments are necessary to determine the optimal concentrations to use. Don't forget that working with salmonella can be hazardous precautions, such as wearing the appropriate personal protective equipment and avoiding the creations of aerosols should be observed when performing this procedure.
