The overall goal of the experiment is to overcome current issues with conventional Annexin five and Propidium iodide staining techniques for evaluation of cell death by apoptosis and or necrosis. We have found that conventional staining approaches of a broad range of cell lines and primary cells results in up to 40%false positive events. These false positive events are due to staining of cytoplasmic RNA.
Conversely, our protocol drastically reduces the number of false positive incidences by introducing fixation and RNA degradation steps at specific stages In the staining procedure, first cells are stained with an X in five and peridium iodide to detect cell death as per conventional procedures. Next, the cells are fixed with 1%formaldehyde, which increases the permeability of the plasma membrane. Finally, the fixed cells are treated with RNAs A in order to degrade cytoplasmic RNA, eliminating false positive causing propidium iodide staining.
Like many cell biologist, we have used conventional nexin five and propidium iodide flow cytometry methods for detecting apoptotic and necrotic cell death. Unfortunately, we have recently found that this method leads to significant number of false positive events up to 40%in a range of cell lines and primary cells. This updated method overcomes these caveats.
Our approach takes advantage of cell fixing and RNA digestion in specific steps throughout the procedure to selectively remove cyop Plasmic RNA, staining that directly leads to these false positive events, Harvest the cells to be analyzed for the experiment. In this video, we use raw macrophages. As an example, centrifuge the samples at 335 Gs for 10 minutes at four degrees Celsius, decant the supernatant and re suspend the cells in two milliliters of one XPBS.
Then wash the cells again under the same conditions. This time, resus suspending the cells in one milliliter of one X NX and five binding buffer. After centrifuging the samples and decanting the supernatant again resuspend the cells in a final volume of 100 microliters of the one X NX Xin five binding buffer.
Add an Xin five to each cell sample according to the manufacturer's recommendations. For example, in this video, 2.5 microliters of an Xin five zi are being added to each polystyrene round bottom tube. Incubate the tubes in the dark for 15 minutes at room temperature.
Then add 100 microliters of one X NX in five binding buffer to each reaction tube. There now should be approximately 200 microliters of solution in each tube. Next, prepare a one to 10 dilution of propidium, iodide or PI in one X NX.In five binding buffer, add four microliters of the diluted PI to each tube, yielding a final concentration of two micrograms per milliliter pi in each sample.
Incubate the tubes in the dark again for 15 minutes at room temperature. After this incubation period, wash the cells with 500 microliters of one x and x and five binding buffer and centrifuge the samples at 335 Gs for 10 minutes at four degrees Celsius and decant the snat. Re suspend the cells in another 500 microliters of one X NX and five binding buffer with 500 microliters of 2%formaldehyde.
To create a 1%formaldehyde solution, mix the tubes by gentle flicking. Fix the samples on ice for 10 minutes after fixing. Add one milliliter of one XPBS to each sample and mix gently by flicking centrifuge the cells at 425 Gs for eight minutes at four degrees Celsius three times during the wash.
Prepare a one to 100 dilution of RNA A in one XPBS, resuspend the pellet by flicking the tubes and add 16 microliters of the diluted RNAs, A solution to give a final concentration of 50 micrograms per milliliter. Then incubate the samples for 15 minutes at 37 degrees Celsius. Next, wash the cells in one milliliter of one XPBS and mix gently by flicking then centrifuge the tubes at 425 GS for eight minutes at four degrees Celsius.
The samples are now ready to be analyzed. Alternatively, the samples can be used for subsequent staining steps if the N-X-M-P-I staining is being performed in parallel with other procedures. Representative image stream flow cytometer images show the extent of false positive staining in three unique cell populations.
One commonly used cell line raw macrophages and two primary cell lines mirroring bone marrow macrophages and goldfish primary kidney macrophages. The use of goldfish primary macrophages highlights the applicability of this technique across a variety of cellular platforms. True positive events display the expected colocalization within the nuclear compartment between PI and drac.
Five, a highly membrane permeable dye that accurately stains the nuclear compartment of living and dead cells as evidenced by the yellow staining. Here, the accuracy of PI nuclear staining was assessed based on its degree of similarity to D dr five BRDU seven A.A D DPI, and DRAC five exhibited greater than 99%similarity in their ability to accurately stain the nucleus in the raw 2 64 0.7 macrophage cell line. By contrast, the cytoplasmic PI staining that occurs when using conventional protocols led to a marked decrease in percent similarity of nuclear staining relative to d dr five.
In this figure, it can be seen that the incorporation of 50 micrograms per milliliter RNAs A removes non-nuclear PI staining and significantly increases the degree of similarity relative to d dr five staining in murn, bone marrow macrophage, and goldfish primary kidney macrophage cells. Representative images of primary kidney macrophages in this figure show the loss of RNA staining in the cytoplasmic compartment with RN a's treatment. These scatter plots of primary goldfish kidney macrophages show a significant reduction of false apoptotic necrotic events in cells prepared with the modified nexin PI protocol.
The new protocol shows that 84.9%of cells are viable. Gates were drawn based on parallel image stream samples that assess fluorescent and morphological features. In one example, early progenitors monocytes and mature macrophages were stained by the modified NX in five PI protocol described in this video, or by the conventional method for this figure cell stained by the modified protocol exhibited a visual reduction in the number of positive PI events due to removal of false positive events by the RNAs treatment.
Further, this effect was more pronounced in large mature macrophage cells than in smaller, early progenitor cells. Conclusions based on conventional protocols would've erroneously suggested a positive correlation in cellular death for the more mature macrophage subsets Following this staining procedure, further steps such as intracellular or surface staining can be performed to answer specific questions about the subpopulations within your cell isolate. The relevance of this technique goes beyond immunity based questions, for example, is also relevant to experimental systems that utilize cells undergoing genotoxic stress cells treated with cell cycle arrest, drugs such as thymidine or hydroxyurea, and studies on embryonic cells where developmental progression is characterized by discrete changes in cellular RNA synthesis.
