The overall goal of this procedure is to detect fluorescent nanoparticle interactions with primary immune cell subpopulations by flow cytometry. This is accomplished by first treating the cells of interest with the nanoparticles. In the next step, the cells are labeled with antibody against cell specific surface markers and then analyzed by flow cytometry.
Ultimately, the interaction of the nanoparticles with individual immune cell populations can be measured. The main advantage of this technique over existing methods like microscopy is that with this technique, variations in fluorescence intensities induced by nanoparticles can be quantified in nonadherent cell populations For nanoparticle internalization in blood leukocytes, or purified monocytes begin by plating five times 10 to the fifth of the cells of interest in one milliliter per well. In each well of a 12 well plate.
Then add 10 microliters of freshly prepared working fluorescent silicon dioxide nanoparticle suspension to each experimental well, adding an equal volume of complete medium to the untreated control wells at this time as well. After a one hour internalization at 37 degrees Celsius, transfer the samples into individual 1.5 milliliter polypropylene tubes, and then spin down the samples for three minutes at 6, 000 times G and room temperature to stain the cells with CD 14 via blue incubate 100 microliters of each cell suspension in 10 microliters of the human antibody at a one to 11 ratio in running buffer for 10 minutes at four degrees Celsius. After washing away the unbound antibody in one milliliter of running buffer resuspend the cells in 200 microliters of fresh running buffer for flow cytometric analysis.
Next, to set up the spectral spillover compensation, open the instrument settings box in the flow cytometry software, then acquire an antibody unlabeled nanoparticle free sample at low fluidic speed. Adjusting the compensation settings in the presence of fluorescent nanoparticles is the trickest part of the procedure as the nanoparticles can interfere with the side scattering to ensure success, a gate of the cell population of interest must be defined Manually adjust the forward and side scattering by regulating the voltage channels. Then draw an appropriately large gate around the desired cell population.
Load another unlabeled sample, and open the compensation tab in the instrument settings box. Adjust the compensation factor during the acquisition until the fluorescence intensity in the spillover channel is roughly the same for the fluorochrome positive and negative populations. Finally, after adjusting the compensation for each single stained fluorochrome control tube, read the nanoparticle treated samples in an effort to better characterize white blood cell behavior in response to nanoparticles, internalization assays were performed as just demonstrated.
For example, in this representative experiment, three major blood leukocytes subpopulations were clearly identified by forward and side scattering after PBMC isolation. Moreover, after fz silicon dioxide treatment, lymphocytes, monocytes, and granulocytes exhibited different nanoparticle internalization rates as indicated by their fluorescence intensity. In these images, nanoparticle internalization is demonstrated in primary CD 14 positive monocytes purified from pbmc.
These scatterplots display CD 14 positive monocytes in the presence of fitz's silicon dioxide nanoparticles. The histogram illustrates the quantification of the fitzy positive cells expressed as fluorescence intensity. Similar internalization experiments were performed with TP one monocytes treated with increasing concentrations of fitz's silicon dioxide nanoparticles with untreated cells as the negative control.
The dot plots illustrate the dose dependent increase in side scattering with an unchanged forward scattering in the TP one cell line after nanoparticle internalization. The data from these graphs suggests that treatment with fitz's silicon dioxide nanoparticles induces a dose dependent internalization in monocytes highlighted by the enhancement of the intracellular granularity to gain further insight into the interactions between immune cells and nanoparticles, microglia were cultured for seven days in vitro and incubated with nanoparticles for one hour. Fluorescence microscopy indicated a mixed primary glial cell culture with a large number of GFP negative non-adherent astrocytes and some GFP positive cells.
In this representative experiment, three, glial subpopulations could be distinguished by flow cytometry with a single CD 11 B antibody staining CD 11 B, negative GFP negative astrocytes and other glial cells microglial CD 11 B positive GFP negative cells, and a CD 11 B positive GFP positive subpopulation. The latter two subpopulations are able to internalize nanoparticles with a slightly increased deficiency by the GFP positive population. Nanoparticle internalization can be further verified by confocal microscopy as demonstrated in this image using rod domine silicon dioxide nanoparticles.
While attempting this procedure, it's important to remember to keep the samples in the dark to avoid bleaching of the fluorescent dyes and to keep the samples at four degrees during the antibody incubation. Following this procedure, other methods like confocal microscopy can be performed to answer additional question about intracellular localization of the nanoparticles.
