Protocol for differential centrifugation-based separation and characterization of apoptotic vesicles derived from human mesenchymal stem cells

Summary Apoptotic vesicles (apoVs) are specific extracellular vesicles generated during apoptosis and play important roles in multiple physiological and pathophysiological settings. Here, we present a protocol using differential centrifugation to separate apoVs from human mesenchymal stem cells (MSCs) after induction of apoptosis. We describe how to characterize apoV size and morphology by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), and determination of specific biomarker expression by immunoblotting. Our protocol will be useful for preparing apoVs for further functional analysis. For complete details on the use and execution of this protocol, please refer to Zheng et al. (2021).


SUMMARY
Apoptotic vesicles (apoVs) are specific extracellular vesicles generated during apoptosis and play important roles in multiple physiological and pathophysiological settings. Here, we present a protocol using differential centrifugation to separate apoVs from human mesenchymal stem cells (MSCs) after induction of apoptosis. We describe how to characterize apoV size and morphology by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), and determination of specific biomarker expression by immunoblotting. Our protocol will be useful for preparing apoVs for further functional analysis. For complete details on the use and execution of this protocol, please refer to Zheng et al. (2021).

BEFORE YOU BEGIN
Each day, billions of cells undergo apoptosis within the body, constituting a crucial activity for homeostasis maintenance (Medina et al., 2020;Morioka et al., 2019). Of note, the generated apoptotic vesicles (apoVs) perform unique functions that canonical extracellular vesicles (EVs), typically released from viable cells, cannot achieve. One of these functions is the delivering of a variety of featured cargos, including DNA, proteins and RNA. For example, apoVs have been found to encapsulate proteins that can regulate apoptosis, such as GSN (Kothakota et al., 1997;Springer et al., 1999). It has also been confirmed that the danger signal protein called HMGB1, is exclusively within apoptotic cell-released EVs (Tucher et al., 2018). In addition, MPO and Arg1, which are contained within apoptotic neutrophil-EVs, may modulate T-cell responses (Shen et al., 2017). Notably, apoVs are becoming a research hotspot for cell-free therapies in a wide spectrum of pathological conditions to restore tissue homeostasis and promote damage repair (Caruso and Poon, 2018;Grant et al., 2019;Kakarla et al., 2020). Here we demonstrate apoV separation protocol from mesenchymal stem cells (MSCs) that emerge as one of the most promising sources for cell therapy (Zheng et al., 2021).

Institutional permissions
The use of human umbilical cord MSCs (hUCMSCs)

MATERIALS AND EQUIPMENT
Note: 10 3 SDS electophoresis buffer can be stored at room temperature (25 C) for up to 3 months.
Note: Before use, the 1 3 SDS electophoresis buffer should be obtained by diluted with ddH 2 O.
Note: 10 3 transfer buffer can be stored at room temperature (25 C) for up to 3 months.
Note: 1 3 transfer buffer can be stored at room temperature (25 C) for up to 2 weeks.

Induction of apoptosis
Timing: 30 min for exchanging the complete culture medium with apoptosis-inducing solution; 12 h for apoptosis induction This major step describes how to induce apoptosis of adherent cells (Figure 1). Given the length of apoptosis induction, we recommend that this step be carried out at the night before conditioned media collection and apoV separation.
CRITICAL: This section should be performed under sterile conditions in a biosafety cabinet.
1. Put the apoptosis-inducing solution and PBS into a 37 C water bath for pre-heating. 2. Aspirate the complete culture medium. 3. Add 5 mL pre-warmed PBS to rinse the cells for 10 s, and then aspirate the PBS.  Note: All rinsing steps should be performed gently so as to avoid cell detachment.
5. Add 5 mL pre-warmed apoptosis-inducing solution to each dish and incubate the cells in a humidified 37 C, 5% CO 2 incubator for 12 h.

Note:
We recommend approximately 12 h of apoptosis induction for MSCs so that $90% cells can undergo apoptosis and release apoVs for separation. Moreover, the maximum duration of apoptosis induction is recommended not to exceed 16 h.
CRITICAL: The duration of apoptosis induction depends on the sensitivity of the cells to the apoptosis-inducing solution. Pre-experiment is needed to determine the induction time length for a specific type of cells.

Separation of apoVs
Timing: 1 h for collecting conditioned media; 5 h for separation of apoVs This major step describes the isolation of apoVs from the conditioned media via differential centrifugation ( Figure 1).
CRITICAL: This section should be performed in a sterile biosafety cabinet.
6. When $90% cells undergo apoptosis (Figure 2), flush the cells thoroughly with pipetting the conditioned media to detach all apoptotic cells.
Note: It is recommended that the cells be observed under an inverted microscope for evaluation of apoptosis. The morphological characteristics of apoptosis include intercellular connection disappearance, cell shrinkage, membrane blebbing and fragmentation into membrane-bound vesicles ( Figure 2). 7. Transfer the supernatant, together with detached cells, to a sterile 15 mL centrifuge tube on ice. 8. Rinse each dish with 2 mL cold PBS, and transfer the PBS to the same centrifuge tube (Each tube contains 7 mL of liquid). 9. Place the centrifuge tubes in a centrifuge. 10. Centrifuge the samples for 10 min at 800 3 g at 4 C to remove the cells and cell debris ( Figure 3A).
Note: All centrifugation steps should be performed at 4 C. The samples and reagents need to be kept on ice.
CRITICAL: Before centrifugation, centrifugal tubes with liquid in the same volume should be placed in a symmetrical position in the centrifuge.
11. Collect the sample supernatant with a pipette and discard the precipitation containing cells and cell debris ( Figure 3A). a. Divide the supernatant into five 1.5 mL EP tubes. b. Place the EP tubes in a centrifuge. c. Centrifuge the tubes at 16,000 3 g for 30 min at 4 C ( Figure 3B).

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Note: When collecting the supernatant, 5 mm supernatant in height above the precipitate can be retained to prevent precipitation from being sucked up.
CRITICAL: The EP tubes should be placed with the cap uniformly oriented towards the center of the rotor, so that the separated apoVs will remain at the bottom of a specific side of the EP tubes.
12. Aspirate the supernatant. a. Add 200 mL cold PBS to each tube and gently resuspend the pellet for sample cleaning by pipetting. b. Merge the suspension of every five EP tubes into one EP tube. c. Centrifuge the tubes at 16,000 3 g for 30 min at 4 C ( Figure 3C).
Note: When removing the supernatant, the EP tubes can be tilted to make the supernatant parallel to the precipitation so as to avoid unnecessary loss of apoVs (Methods video S1). a. Add 1 mL cold PBS to each tube and gently resuspend the pellet for sample cleaning by pipetting. b. Centrifuge the tubes at 16,000 3 g for 30 min at 4 C. 14. Aspirate the supernatant, and re-suspend the precipitate with the corresponding solvent ( Figure 3C). The precipitation is the apoVs. (If the yield of apoVs is low or unstable please read troubleshooting Problem 1-2). a. Re-suspend the precipitate separated from one dish with 50 mL cold PBS and store at 4 C for nanoparticle tracking analysis (NTA) analysis. b. Re-suspend the precipitate separated from one dish with 50 mL cold PBS and store at 4 C for TEM analysis. c. Re-suspend the precipitates separated from two dishes with 50 mL lysis buffer, incubate on the ice for 30 min and store at À80 C for Western blotting (WB) analysis.

Characterization of apoVs: Nanoparticle tracking analysis
Timing: 3 h

Note:
The NTA protocol below is specifically for the microscope model PMX-120. NTA measurement using different machine will need different protocol.
NTA is used for measuring the amount and size of apoVs by capturing the movies of apoV particles that move under Brownian motion (Figure 1).
15. Take 10 mL of the 50 mL sample, and dilute to 10 mL with PBS.
Note: The samples should be maintained on ice during transportation.
16. Use a sterile 10 mL syringe to inject at least 5 mL ddH 2 O to flush the ZetaViewâ BASIC NTA machine channel at a constant speed (0.5 mL/s to 1 mL/s), until the number of particles displayed on the detection interface is less than 10 (Methods video S2). 17. Prepare calibration solution (diluted at 1:250,000).
a. Dilute 1 mL calibration solution provided with the NTA machine to 1 mL with ddH 2 O. b. Take 100 mL and dilute to 25 mL with ddH 2 O.
Note: The calibration solution needs to be freshly prepared immediately before use.
18. Use a sterile 10 mL syringe to inject at least 5 mL calibration solution to flush the machine channel (Methods video S3).
Note: Maintain a uniform and stable flow during the injection process so as to avoid the formation of bubbles.
19. When the number of particles displayed on the detection interface is between 50-400 (preferably around 200), run the calibration program (Methods video S4). 20. Repeat step 16 to clean the machine channel (Methods video S5). 21. Use a sterile 10 mL syringe to inject at least 5 mL buffer solution (PBS) to flush the machine channel at a constant speed (0.5 mL/s to 1 mL/s), until the number of particles displayed on the detection interface is less than 10 (Methods video S6). 22. Slowly inject the sample into the machine channel (at least 2 mL) using a sterile 1 mL syringe (Methods video S7).
Note: Before injection, hold the syringe upright and tap the side gently to drain the bubbles in the sample.

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Note: For samples with an optimum particle concentration, the number of particles displayed on the detection interface should be between 50-400 (preferably around 200).
CRITICAL: If the reading is too high, repeat step 16 immediately to avoid particles retention at the channel wall. Use PBS to further dilute the sample, and when the number of particles displayed on the detection interface is less than 10, repeat step 21-22.
23. Conduct particle capture analysis and generate analysis report. (If contaminants are found in NTA please read troubleshooting Problem 3). 24. Repeat step 21 to clean the sample in the channel. 25. Repeat step 16 to clean the PBS in the channel. 26. Use a sterile 10 mL syringe to inject at least 10 mL air at a constant speed (0.5 mL/s to 1 mL/s) to remove the ddH 2 O in the channel.
Note: If multiple samples need to be analyzed, carry out step 20-23 above in sequence.

Characterization of apoVs: Transmission electron microscope analysis
Timing: 4 h TEM is used for analyzing the size and morphology of apoVs (Figure 1).
27. Drop 10 mL of the 50 mL sample onto a copper mesh and incubate for 10 min at 25 C. Remove excess liquid with filter paper.
Note: In order to ensure that single particles can be observed, the sample concentration should be adjusted and the suspension should be clear without obvious turbidity. The sample used here is diluted at 1:1,000. However, the dilution of different cells should be determined by the preliminary experiment.
Note: Before dropping, mix the suspension well and pipette from the middle of the liquid.
CRITICAL: Pay attention to dropping the sample to the front side of the copper mesh.
28. Rinse the copper mesh in 100 mL sterile ddH 2 O and remove excess liquid with filter paper. Repeat this step for another two times.
Note: Use electron microscope tweezers to clamp the edge of the copper mesh, and immerse the front side downward into ddH 2 O to rinse it.
29. Negatively stain the copper mesh by immersing the front side in 2% uranyl acetate solution for 1 min at 25 C, and remove the excess liquid with filter paper. 30. Repeat step 28. 31. Remove excess liquid with filter paper and put the copper mesh on a new filter paper to dry it under an incandescent lamp for 2 min or at room temperature (25 C) for 10 min. 32. Observe the copper mesh under a TEM operating at 100 kV and imaged with a digital camera. (If contaminants are found in TEM analysis please read troubleshooting Problem 3.) (If the morphology of apoVs is not typical please read troubleshooting Problem 4). The protein concentration of apoVs is quantified via the BCA assay and the expression levels of specific markers for apoVs are detected via WB (Figure 1).

Characterization of apoVs: Immunoblotting analysis
Note: Methods for determination of protein concentration can be selected according to laboratory conditions, such as using Qubit protein quantification assay . This protocol only provides a relatively simple and common determination method.
33. Centrifuge the lysates (the sample obtained at step 14c) at 13,000 3 g for 5 min at 4 C. 34. Transfer the supernatant to a sterile 1.5 mL EP tube.
Note: Place all the samples on the ice during the operation.
35. Preparation of 0.5 mg/mL BSA standard solution. 36. Dilute 0.5 mg/mL BSA standard solution with normal saline to a serial concentration. 37. Add 20 mL of standard solution at different concentrations into each standard well of the 96-well plate, and add 2 mL sample and 18 mL normal saline into each sample well of the 96-well plate.
Note: Be careful when adding to the microplate so as to avoid the formation of bubbles.
38. Mix BCA reagent A and BCA reagent B in a 50 : 1 ratio to make the needed amount of working solution.
Note: It is recommended that the working solution be prepared right before use.
39. Add 200 mL working solution to each well, and incubate the 96-well plate in an incubator at 37 C for 30 min. 40. Put the 96-well plate into the microplate reader and measure the optical density (OD) value at the wavelength of 562 nm. 41. Establish the standard curve according to the OD data and calculate the protein concentration of each sample. 42. According to the results, dilute the samples to 1 mg/mL with normal saline and 5 3 loading buffer. 43. Seal the tubes with parafilm and heat them at 95 C for 5 min. 44. Load equal protein mass per sample onto a precast 4%-20% (wt/vol) TGX stain-free gel.
Note: Choose the gel percentage according to the molecular weight of the proteins of interest.
45. Run the SDS-PAGE at 80 V for the concentrated gel and 120 V for the separation gel.
Note: The purpose of this step is to resolve the proteins based on their sizes.
Note: The running time may vary according to the equipment or percentage of gel.
46. Transfer the gel to a polyvinylidene difluoride (PVDF) membrane using a wet transfer system for 120 min at 200 mA.
Note: The PVDF membrane needs to be rinsed shortly (20 s) in methanol immediately before transferring.
47. Block the membrane in 1 3 TBST containing 5% BSA for 2 h at 25 C with gentle shaking.
Note: The 1 3 TBST is obtained by diluting the 10 3 TBST with ddH 2 O.

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48. Incubate the membrane with the primary antibodies diluted in 1 3 TBST at 4 C for 12-16 h with gentle shaking. a. The anti-Caspase-3 antibody that detects both full length Caspase-3 and Cleaved Caspase 3 is used at a 1:1,000 dilution as specific markers for apoptotic products (Nicholson et al., 1995). 49. Wash the membrane in 1 3 TBST for 10 min at 25 C on a shaker. Repeat this step for another two times. 50. Incubate the membrane with the secondary antibodies diluted in 1 3 TBST for 1 h at 25 C on a shaker. 51. Wash the membrane in 1 3 TBST for 10 min at 25 C on a shaker. Repeat this step for another two times. 52. Incubate the membrane with the reagents of an ECL kit for 1 min away from light. 53. Detect the protein bands by a gel imaging system. (If low signal or no signal of Cleaved Caspase-3 is detected by immunoblotting please read troubleshooting problem 5).

EXPECTED OUTCOMES
Here, we provide a detailed protocol for the separation and characterization of apoVs produced by MSCs. From our experience, this approach typically yields about 30 mg apoVs/100-mm dish. The size distribution of apoVs analyzed by NTA shows that the diameter range of apoVs is 50-500 nm, with majority peak at 100-200 nm ( Figure 4). Besides, the morphological characteristics of apoVs as cupshaped membranous vesicles (Zheng et al., 2021) are verified by TEM observation (Figure 5). Moreover, immunoblotting analysis demonstrates that Cleaved Caspase-3, a typical apoptosis marker (Nagata, 2018), accumulates in apoVs, with expression of plasma membrane markers, Caveolin-1 and Flotillin (Durcin et al., 2017), and the cytoskeleton marker, b-actin, but without detection of the nuclear envelop protein, Lamin B1, as a negative control (Figure 6).

LIMITATIONS
The yield and quality of apoVs depends largely on the apoptosis inducing process. Since the sensitivity to apoptosis inducer varies among different cell types, the precise induction concentration and induction time need to be confirmed by pre-experiment. Moreover, during the apoptosis inducing process, the researcher needs to closely observe the morphological changes of cells in order to verify the appropriate timepoint for collecting conditioned media. It should be noted that excessive apoptosis can lead to secondary necrosis (Rogers et al., 2017;Silva, 2010), in which the cell membrane breaks down and the contents are released, which will contaminate the separated apoVs. The apoptosis versus secondary necrosis can be distinguished by Annexin V/PI or 7-AAD staining, if needed (Akiyama et al., 2012).
Centrifugation at 800 g for 10 min applied in this study removes most of the larger contaminants including cells and cell debris, but also results in loss of large apoVs. However, this protocol is advantageous on the separation of small apoVs (50-400 nm) from cultured cells without ultracentrifugation, which is thus simple, convenient and feasible for subsequent morphological, functional and therapeutic studies (Zheng et al., 2021). Notable, for apoVs separated from tissue or body fluid samples, the centrifugal speed or separation method must be adjusted according to the component complexity of these samples and the size of target apoVs in practical operation .
The purity of apoVs separated by this differential centrifugation approach is relatively low, due to the aggregation of EVs as well as contaminating proteins (Yang et al., 2020). Besides, the subpopulation of apoVs cannot be distinguished (Bordanaba-Florit et al., 2021). To further separate the apoVs subclasses, it is recommended that this approach be performed in combination with other methods such as immunoaffinity separation (Koliha et al., 2016) and high-sensitivity fluorescence-activated sorting (Kondratov et al., 2020).
The current protocol has been optimized for adherent cells. As for separating apoVs from suspension cells, when transferring the complete culture medium to apoptosis-inducing solution, the suspension cells need to be initially collected via extra-centrifugation step (Dou et al., 2020).

Potential solution
Scaling up the cell number by preparing more plates will increase the overall yield of apoVs (''cell culture''). Besides, the apoptosis condition is a critical factor for apoV yield. Thus, the concentration of apoptosis-inducing solution and the length of induction time needs to be optimized via preexperiment (steps 5 and 6).

Problem 2
The yield of apoVs is unstable (step 14).

Potential solution
The density and condition of cells when inducing apoptosis is important for the amount of released apoVs. We recommend cells at early passages be used and the procedure start when cells reach 80%-90% confluency (''cell culture'').

Problem 3
Contaminants in TEM and NTA (steps 23 and 32).

Potential solution
In the characterization of apoVs, the cleanliness of PBS is also crucial. We suggest making it on the spot. Or use commercial PBS within 1 week of unsealing.

Problem 4
The morphology of apoVs is not typical when observed via TEM (step 32).

Potential solution
The concentration of apoV sample is crucial for the TEM analysis. Thus, adjust the sample concentration so as to make it within an appropriate range (step 27). In addition, since long-term storage will affect the morphology of EVs, it is recommended to perform TEM observation within a short time after apoV separation (step 14) (Gelibter et al., 2022).

Problem 5
Low signal or no signal of Cleaved Caspase-3 is detected by immunoblotting (step 53).

Potential solution
As has been stated above, the concentration of apoptosis-inducing solution and the length of induction time are critical for the efficiency of apoV secretion (step 5). Before the collection of conditioned media, the cells should be observed under an inverted microscope for evaluation of apoptosis (step 6). In addition, poor signal may result from problems during the WB process. We recommend using fresh primary and secondary antibodies, increasing the concentration of primary antibody and extending the incubation time (steps 48 and 50).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yan Jin (yanjin@fmmu.edu.cn).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate/analyze any dataset or code.