The overall goal of this protocol is to both detect and isolate apoptotic bodies to high purity. Apoptotic bodies are a type of extracellular vesicles generated to help the induction of hematosis. They are the largest type of extracellular physical family typically ranging from one to five micrometer diameter.
It is now becoming apparent that apoptotic bodies can aid cellular processes, such as apoptotic cell clearance and intracellular communication. Therefore, the development of an accurate methodology to detect, quantify, and isolate apoptotic bodies is critical for the personalization of their functions and mechanisms. In this series of perparedments we're gonna show you how to detect and quantify and isolate apoptotic bodies from apoptotic samples.
Part A, the induction of apoptosis. To induce apoptosis in cell lines, such as THB1 monocytes, first start by collecting the cells. Count cells and collect approximately 10 million cells or as required.
We recommend 10 million for analysis of apoptotic bodies. Alloquate two million cells per well of a six well plate. Remove the lid from the six well plate and UV cells using the UV Stratalinker 1800.
Irradiate cells at 150 millijules per centimeter squared. This irradiation should take approximately 30 to 60 seconds. After the irradiation is complete, incubate cells for two to eight hours, depending on the cell line.
At this point, cells should exhibit apoptotic morphologies, such as apoptotic blemming and apoptotic body formation. Collect apoptotic cells with a P1000 pipette. Collect approximately 1/10th of the apoptotic sample for the whole apoptotic sample control.
Also collect an untreated or viable sample. Centrifuge both the whole apoptotic sample and untreated sample at 3, 000g for six minutes. Re suspend in PBS and set aside on ice.
For apoptotic body isolation, continue to either step B or C.B, Apoptotic body isolation using FACS. Centrifuge the remaining apoptotic sample at 3, 000g for six minutes. Remove the supernatant from the remaining apoptotic sample.
Re suspend the remaining apoptotic sample in staining solution containing the nexum 550 and topo three staining for 10 minutes, at room temperature, in the dark. After centrifugation, re suspend in FACS buffer and then filter through a 70 micron cell strainer to ensure cells are efficiently separated. Perform a standard setup of the FACS machine.
Acquire the whole apoptotic sample and change the voltage settings to ensure that all events are within the FACS bots and populations can be easily separated. Setup the gating strategy as described in the flow cytometry gating strategy section later. Select gated apoptotic body population and the desired number of apoptotic bodies for collection.
Perform an initial sort to confirm the sort settings and apoptotic body purity. After a sort has been performed, perform a system black flush and reanalyze the sorted sample. If high purity is achieved, continue to sort the desired number of apoptotic bodies.
Once sorting is complete, take a small portion of the pro sort apoptotic body sample and reanalyze to confirm purity. Part C, apoptotic body isolation via differential centrifugation. Centrifuge the remaining apoptotic sample at 300g for 10 minutes.
This centrifugation step will separate cells, including apoptotic cells, viable cells, and necrotic cells from small extracellular vesicles, which will remain in the supernatant. In a new falcon tube, collect the supernatant containing the extracellular vesicles. Re suspend the apoptotic enriched faction in PBS and set aside on ice with the untreated and whole apoptotic samples.
Centrifuge the collected supernatant containing the apoptotic bodies at 3, 000g for 20 minutes. Remove the supernatant containing the smaller, extracellular vesicles, being careful to not disrupt the pellet which will contain the apoptotic bodies. Re suspend the apoptotic bodies in PBS and collect 100 microliters of each the untreated, whole apoptotic sample, the apoptotic cells enriched, and the apoptotic bodies enriched sample and stain with the nexum five and topo three staining solution.
Stand in the dark for 10 minutes at room temperature before performing flow cytometry analysis. Analyze as described in the laid out gating strategy. Part D, flow cytometry gating strategy.
To begin flow cytometry analysis, separate topo three high necrotic cells from all other events by comparing topo three and four together. From the non-permeabilization events, perform the next gate. Select the non-permeabilization events and compare a side scatter verse a nexum five.
Select two populations, including a side scatter intermediate in high, a nexum five low to intermediate cells. This population is P1.Also create a second population including all other events. This population is P2.P1 will contain viable cells and early apoptotic cells.
P2 will contain all apoptotic events and debris. Select P2 for further gating. From P2, remove all debris by comparing topo three verse a nexum five.
Select all of nexum five intermediate and high events. This population will now include all apoptotic bodies and apoptotic cells. Select this population.
To separate apoptotic bodies from apoptotic cells, compare full scatter to the nexum five. To select apoptotic bodies, select full scatter low. To select apoptotic cells, select full scatter intermediate to high events.
When performing apoptotic body isolation by FACS we recommend performing one final gating strategy by selecting the apoptotic body fraction and viewing it by topo three verse a nexum five. Select all events. This is used to select the gating strategy based on florescence rather than full scatter and side scatter properties to increase accuracy for sorting.
To identify viable cells, go back to P1 population. For general viable cell analysis, select P1 and compare full scatter to the nexum five. Select all full scatter intermediate to high cells.
This will include all viable cells, removing any remaining debris. Alternatively, for more in depth apoptosis analysis, viable and early apoptotic cells can be separated. For this, compare topo three verse full scatter.
Viable cells will be topo three low, full scatter intermediate to high. Early apoptotic cells contain intermediate topo three staining. This entire gating strategy can then be applied to all samples being analyzed.
For example, for the apoptotic bodies isolated after FACS. Apply the gating strategy to all samples. Apoptotic body purity can then be prepared.
For example, by selecting only the nexum five positive populations, comparing full scatter and a nexum five. This therefore demonstrates the apoptotic bodies post-isolation. Representative results.
Flow cytometry analysis was performed to confirm the induction of apoptosis. This gating strategy allows the identification of viable cells, early apoptotic cells, late apoptotic cells, and necrotic cells in the UV irradiated sample. This methodology demonstrated the isolation of apoptotic bodies by two approaches.
Firstly, a FACS-based approach, where apoptotic bodies were enriched to 99%purity and a differential centrifugation approach where apoptotic bodies were isolated to 97%purity. This was confirmed by flow cytometry where apoptotic bodies were separated by cells, including viable cells, apoptotic cells, and necrotic.Conclusion. Our approach may provide a new tool for apoptosis and apoptotic body research.
Therefore contributing to their advances in personalization of these mechanisms and to help produce new developments in targeting these processes.
