Flow Cytometry Analysis of Planarian Stem Cells Using DNA and Mitochondrial Dyes

Mohamed  Mohamed Haroon; Praveen Kumar Vemula; Dasaradhi  Palakodeti

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May 2021

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Abstract
Background
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
Competing interests
References

Flow Cytometry Analysis of Planarian Stem Cells Using DNA and Mitochondrial Dyes
利用DNA和线粒体染料流式细胞术分析涡虫干细胞

Mohamed Mohamed Haroon*

Praveen Kumar Vemula* Dasaradhi Palakodeti

Dasaradhi Palakodeti* (*共同第一作者)

DOI: 10.21769/BioProtoc.4299

卷期号： Vol 12, Iss 2, January 20, 2022

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Abstract

Planarians are free-living flatworms that emerged as a crucial model system to understand regeneration and stem cell biology. The ability to purify neoblasts, the adult stem cell population of planaria, through fluorescence-activated cell sorting (FACS) has tremendously increased our understanding of pluripotency, specialization, and heterogeneity. To date, the FACS-based purification methods for neoblasts relied on nuclear dyes that discriminate proliferating cells (>2N), as neoblasts are the only dividing somatic cells. However, this method does not distinguish the functional states within the neoblast population. Our work has shown that among the neoblasts, the pluripotent stem cells (PSCs) are associated with low mitochondrial content and this property could be leveraged for purification of the PSC-enriched population. Using the mitochondrial dye MitoTracker Green (MTG) and the nuclear dye SiR-DNA, we have described a method for isolation of PSCs that are viable and compatible with downstream experiments, such as transplantation and cell culture. In this protocol, we provide a detailed description for sample preparation and FACS gating for neoblast isolation in planaria.

Keywords: Planaria (涡虫), Regeneration (再生), Stem cells (干细胞), Pluripotency (多能性), FACS (FACS), Mitochondria (线粒体)

Background

Fluorescent dyes that stain the nucleus in live cells are used for fluorescence-activated cell sorting (FACS) proliferating stem cells from planaria. In a seminal work, Hayashi et al. used Hoechst for staining planarian cells and categorized the cells into three populations, termed as X1, X2, and Xins population (Figure 2) and (Hayashi et al., 2006). This allowed sorting of the proliferating cells (X1 cells) in the planarian field of research (Hayashi et al., 2006). Later, Hoechst stain was found to be toxic so, for downstream applications where viable cells are required, a different FACS gate X1(fs) based on forward scatter (FSC) had to be used (Wagner et al., 2011). This population was obtained by back-gating the X1 population in an FSC vs SSC plot (Wang et al., 2018). However, X1(fs) is heavily contaminated by differentiated cells. Subsequently, the nuclear dye SiR-DNA was proposed as an alternative to Hoechst for obtaining proliferating stem cells with high viability (Lei et al., 2019; Niu et al., 2021). However, this flow cytometry method using a nuclear dye could not distinguish between the pluripotent and the specialized stem cell populations.

Methods that could distinguish pluripotent from specialized stem cells could advance our understanding of planarian stem cell biology and function. Since neoblasts have scant cytoplasm and reduced organelles, we reasoned that organelle complexity could be way to distinguish different cellular states (Higuchi et al., 2007). For this purpose, we used mitochondria, an organelle that plays an important role in metabolism and signaling. Studies in mammalian stem cells have suggested that the mitochondrial state, including the mitochondrial activity, content, morphology, and number varies between different cellular states. We have recently reported the use of MitoTracker Green (MTG) staining in combination with nuclear dyes, such as Hoechst and SiR-DNA, for the purification of pluripotent stem cells (Mohamed Haroon et al., 2021). Our data suggest that the population of proliferating cells with low MTG is enriched in pluripotent cells compared to the high MTG cells, as indicated by higher transplantation efficiency (~65% for MTG Low cells vs ~30% for MTG High cells). Further, this method could be used to enrich the 2N stem cells from the X2 population, which is a mixture of stem cells and mitotic progenitors. To this end, the X2 gate was divided into four populations based on MTG and FSC, which indicates size. We found that the 2N PSCs are enriched in the low MTG and high FSC gate (Mohamed Haroon et al., 2021). In contrast, X2 cells with low MTG and low FSC were predominantly early post-mitotic progenitors. The X2 high MTG cells, regardless of their size, were observed to be late progenitors (Mohamed Haroon et al., 2021). Here, we provide a step-by-step protocol for planarian dissociation, staining, and FACS gating of various MTG populations.

Materials and Reagents

50 mL centrifuge tubes (Tarsons, catalog number: 546021)
15 mL centrifuge tubes (Tarsons, catalog number: 546041)
35 mm culture dish
60 mm culture dish
Falcon 5 mL round bottom FACS tubes (BD, Falcon, catalog number: 352054)
Sphero^TM Rainbow fluorescent particles (BD Biosciences, catalog number: 556291)
BD FACS Accudrop beads (BD, catalog number: 345249)
0.45 μm Millex-HV syringe filter unit (Merck Millipore, catalog number: SLHVR33RS)
0.22 μm Millex-GV syringe filter unit (Merck Millipore, catalog number: SLGVR33RS)
40 µm cell strainer (BD Falcon, catalog number: 352340)
Surgical blade No. 23 (Lister)
Bovine serum albumin (Sigma-Aldrich, catalog number: A2153)
Hoechst 33342 (Invitrogen, catalog number: H3570)
MitoTracker Green FM (Invitrogen, catalog number: M7514)
SiR-DNA (Cytoskeleton, Inc. catalog number: CY-SC007)
DAPI (Sigma-Aldrich, catalog number: D9542)
Propidium iodide (Sigma-Aldrich, catalog number: P4864)
MEM essential amino acids solution (50×) (ThermoFisher Scientific, Gibco^TM, catalog number: 11130051)
MEM non-essential amino acids solution (100×) (ThermoFisher Scientific, Gibco^TM, catalog number: 11140050)
MEM Vitamine solution (100×) (ThermoFisher Scientific, Gibco^TM, catalog number: 11120052)
Sodium pyruvate (100×) (ThermoFisher Scientific, Gibco^TM, catalog number: 11360070)
100× Penicillin streptomycin (ThermoFisher Scientific, Gibco^TM, catalog number: 15070063)
L-glutamine (ThermoFisher Scientific, Gibco^TM, catalog number: 25030081)
Fetal bovine serum (Himedia, catalog number: RM9955)
NaCl
CaC₃
MgSO₄
MgCl₂
KCl
NaHCO₃
NaH₂PO₄
KCl
Glucose
HEPES (free acid)
HEPES (sodium salt)
MnCl₂
KH₂PO₄
D-biotin
D-glucose
D-trehalose
Tricine
Montjuïc salts (see Recipes)
Calcium, magnesium free buffer with BSA (CMFB) (see Recipes)
IPM + 10% FBS (see Recipes)

Equipment

Refrigerated centrifuge (Eppendorf, model: 5810R)
FACS sorter (BD ARIA III or BD ARIA Fusion)

Software

FlowJo
BD FACS DIVA

Procedure

Preparation of cell suspension
1. Separate the required number of worms that have been starved for at least 7 days and wash with fresh 1× Montjuïc salts.
  
  Note: This protocol is optimized with the planarian species, Schmidtea mediterranea, and both sexual and asexual biotypes can be used. The number of worms required depends on the number of cells required for the downstream applications. In our experience, 35 worms of ~0.7 cm size will yield ~100,000 to 150,000 X1 cells.
2. Transfer the worms to the cap of a 60 mm or 35 mm culture dish. Remove the 1× Montjuïc salts completely.
3. Immediately add ~100 μL of CMFB to the planarians (Figure 1A).
  
  Note: The volume of CMFB is approximate, as it should be enough to cover all the planarians. The volume could be scaled up or down based upon the specific requirement.
4. Using a scalpel, slice the worms into pieces as small as possible (Figure 1B). Intermittently wipe the scalpel with tissue paper, to remove the mucous. Ensure that no large fragments remain.
5. Once the tissues are diced into small fragments, add 1 mL of CMFB.
6. Make a wide bore 1 mL pipette tip by cutting its end using a sterile scalpel (Figure 1C). Using this wide-bore pipette, transfer the tissue fragments to a fresh 50 mL centrifuge tube.
  
  Note: The tissue fragments may stick to the surface. If so, flush the tissue with excess CMFB and transfer it to the centrifuge tube. In our lab, we take the tissue fragments in ~5 mL of CMFB.
7. Mechanically dissociate the cells using a 1 mL pipette by gently pipetting multiple times. After ~20-30 times of pipetting, let the centrifuge tube sit for ~2-3 min. The undissociated tissue fragments will settle down. Transfer the supernatant containing cells to a fresh 50 mL tube without disturbing the settled tissue fragments. Leave some supernatant, so that the undissociated fragments don’t come along with the supernatant (Figure 1D).
8. Add another 3-4 mL of CMFB solution to the undissociated fragments and dissociate again by gently pipetting.
9. Repeat Steps A7 and A8 till all the tissue fragments are completely dissociated.
  
  Note: Steps A7 and A8 are to reduce cell death as much as possible, since pipetting of already dissociated cells is unnecessary and may induce cell death.
10. Collect all the cell suspensions and strain through a 40 μm strainer into a fresh 50 mL centrifuge tube.
11. Centrifuge the strained cell suspension at 290 × g at 4°C for 10 min in a swinging bucket rotor. A dark cell pellet is observed at this stage (Figure 1E).
12. Discard the supernatant and resuspend the cell pellet in IPM + 10% FBS media. The volume of media depends on the number and size of the worms used. For ~40 worms of ~0.7 cm, 10 mL of media could be used.
  
  Figure 1. Preparation of cell suspensions.
  
  A. Planarians are taken in the cap of a 35 mm culture dish and ~100 μL of CMFB is added. B. Representative image of diced planarian fragments. C. Representative image of the 1 mL pipette with the tips cut (right) compared to a normal pipette (left). D. After mechanical disruption by pipetting the cell suspension ~20-30 times, the solution is allowed to settle down (left centrifuge tube) and the supernatant is transferred to a fresh centrifuge tube (right) and fresh CMFB is added to the undissociated fragments. See Steps A7-A9 for details. E. Cell pellet after cell straining and centrifugation.
Staining of the cell suspensions
1. Add 40 μg/mL Hoechst 33342 to the cell suspension and incubate at room temperature in the dark for 40 min. Mix the solution intermittently at an interval of ~10 min.
2. After 40 min, add 100 nM MTG to the same cell suspension and incubate for another 20 min at room temperature in the dark.
3. Post incubation with MTG, centrifuge the cell suspension at 290 × g for 10 min at 4°C in a swinging bucket rotor. Discard the supernatant and resuspend the cell pellet in IPM + 10% FBS media.
  
  The final volume could be adjusted based on the efficiency of the sorting machine. In our laboratory, for ~40 worms of ~0.7 cm, we use 3 mL of IPM + 10% FBS media.
4. Finally, add 1 µg/mL of propidium iodide to the cell suspension to discriminate live/dead cells.
5. For FACS sorting of cells for transplantation or in vitro culture experiments, SiR-DNA (1 μM) should be used instead of Hoechst 33342 at Step B1. At Step B4, for live/dead cell discrimination, use 10 μg/mL of DAPI instead of propidium iodide. The rest of the staining protocol remains the same.
FACS gating for X1 and X2 MTG populations
1. For sorting, use a 100 μm tip at 20 psi sheath pressure. Use the proprietary BD Sheath fluid.
  
  Note: Before every sorting session, we routinely calibrate the instrument by setting up the appropriate laser delay with the Sphero^TM Rainbow fluorescent particles and the drop delay with the BD FACS Accudrop beads. This step should be done according to the instrument used and the information is generally available with the instrument manual.
2. Run the Hoechst 33342 and MTG stained cells in the FACS machine. Adjust the forward scatter (FSC) and side scatter (SSC) voltage and make a gate (P1) to exclude debris and dead cells.
3. Next, gate the singlets using an FSC A (area) vs FSC W (width) dot plot (P2).
4. Within this singlet population, select the live cells indicated as propidium iodide negative events (P3).
5. Set the gates for X1, X2, and Xins population as represented in Figure 2 and Mohamed Haroon et al. (2021).
  
  Figure 2. Representative FACS gating for X1, X2, and Xins populations.
  
  See Steps C2-C5 for details. FSC- forward scatter, SSC- side scatter, FSC-W width, FSC-A area. The blue (Hoechst Blue) emission of Hoechst 33342 can be collected using a 450/20 bandpass filter and for the red emission (Hoechst red), use 630/40 bandpass filter.
6. To obtain a population with enriched pluripotent cells, gate MTG Low cells within X1. Similarly to enrich the lineage primed population gate corresponding to MTG High within the X1 cells (Figure 3A).
7. The X2 gate could also be separated into MTG Low and MTG High cells. To enrich the 2N stem cells within X2, make four gates based on MTG and FSC parameters (Figure 3B).
  
  Note: C6-C7: With the current understanding, it is impossible to mark the exact fluorescent intensity or percentage of MTG Low cells that exactly represent stem cells while excluding the differentiating progenies in both X1 and X2 populations. Hence, for consistency, the population can be divided into equal percentages of MTG Low and MTG High.
  
  Figure 3. Gating strategy for MitoTracker Green (MTG) populations within (A) X1 and (B) X2 cells.
  
  For consistency, an equal percentage of MTG Low and High populations should be gated. HFSC-High FSC, LFSC- Low FSC.
FACS gating for SiR-DNA 2N and 4N MTG populations
1. Run SiR-DNA stained cells and set gates P1 and P2 as described above, to eliminate debris and doublets. Next, gate DAPI negative cells (P3) to obtain the live cells.
2. Within the live cells, gate SiR-DNA 2N (P4) and 4N (P5) populations (Figure 4).
  
  Note: Unlike Hoechst staining, SiR-DNA staining does not separate 2N and 4N cells well in a FACS plot. Hence, to ensure accurate gating, use the reference plots in Figure 4 and other Sir-DNA plots (Lei et al., 2019). Alternatively, one could use 6000 rads irradiated animals (>2 days post irradiated animals) as a control that is devoid of proliferating stem cells (Wagner et al., 2011).
  
  Figure 4. Representative FACS gating for 2N (P4-gate) and 4N (P5-gate) cells using SiR-DNA nuclear dye. See Steps D1-D2 for details.
3. To obtain the population that is X1 equivalent, first determine X1(fs) gate by back-gating X1 cells in an FSC vs SSC plot (Figure 5) (Wagner et al., 2011). Within the X1(fs) population, gate the SiR-DNA 4N population as determined in the earlier Step D2 (Figure 5).
4. Similarly, determine X2(fs) gate by back-gating X2 cells in FSC vs SSC plot, and gate the SiR-DNA 2N population within the X2(fs) gate (Figure 5).
5. Set gates for MTG Low and High, as well as FSC low and high within 4N and 2N gates, similarly to the Hoechst stained cells as explained in Steps C7-C8.
  
  Figure 5. Representative FACS graph for sorting 2N and 4N SiR-DNA population within X2(fs) and X1(fs) gates.
  
  This gating is performed to reduce cross-contamination of X1 cells and X2 cells, as the SiR-DNA dye could not resolve the cell cycle stages well, unlike Hoechst.
FACS sorting
1. Sort the cells in 5 mL FACS tubes containing 2 mL of IPM + 10% FBS media. Keep the collection chamber at 4°C using the appropriate adaptor.
  
  Note: We routinely perform the sorting using the ‘purity’ mode described in BD FACS Aria III and BD FACS Aria Fusion sorters. Such sort precision modes are instrument specific, and the users should refer to the instrument manual for the appropriate settings. The users can run the sorted samples in the FACS machine to ensure the purity of the sort. In addition, fluorescent in situ hybridization or immunostaining for neoblast markers, such as piwi-1, can be performed to check the purity of the sorted samples.
2. Centrifuge the sorted cells at 300 × g for 10 min at 4°C using a swinging bucket rotor. Discard the supernatant and resuspend the cells in an appropriate volume based on the downstream applications.
  
  Note: The cell pellet after sorting ~100,000 to 150,000 cells will be very small and may not be readily visible.

Data analysis

The flow-cytometry graphs are analyzed using the BD FACS DIVA software. Alternate software such as FlowJo could also be used. The isolated cell populations were characterized by PIWI-1 marker expression and transplantation experiments over a minimum of three independent replicates (Mohamed Haroon et al., 2021). Statistical analysis were performed using an unpaired, two-tailed Student’s t-test.

Recipes

1× Montjuïc salts

NaCl 1.6 mM

CaC₃ 1 mM

MgSO₄ 1 mM

MgCl₂ 0.1 mM

KCl 0.1 mM

NaHCO₃ 1.2 mM

pH 7.0
Calcium, magnesium free buffer with BSA (CMFB) (For 1,000 mL)

NaH₂PO₄ 400 mg

NaCl 800 mg

KCl 1,200 mg

NaHCO₃ 800 mg

Note: 10× concentration of the above salts could be prepared (filtered and autoclaved) separately and stored as stock.

Glucose 240 mg

HEPES 15 mM

pH 7.3

BSA 1%

Note: 1% BSA should be added fresh to the buffer.
IPM + 10 % FBS (For 1,000 mL)

HEPES (free acid) 7,208.8 mg

HEPES (sodium salt) 3,514.1 mg

NaCl 985.4 mg

NaHCO₃ 800.1 mg

KCl 26.4 mg

CaCl₂ 113.23 mg

MgSO₄ 44.02 mg

MnCl₂ 0.19 mg

KH₂PO₄ 68.5 mg

D-biotin 0.3 mg

D-glucose 300 mg

D-trehalose 50 mg

Tricine 2.5 mg

MEM Essential amino acids 2 mL

MEM non-essential amino acids 5 mL

MEM vitamin solution 3 mL

Sodium pyruvate 14 mL

Penicillin/streptomycin 10 mL

L-Glutamine 2 mL

Fetal Bovine Serum 100 mL

pH 7.3

Acknowledgments

The protocol is majorly adapted from the recently published work from our laboratory (Mohamed Haroon et al., 2021). The authors thank Central Imaging and Flow Cytometry Facility (CIFF-BLiSc campus) for the state-of-the-art flow cytometry facility. MMH thanks DBT for funding the graduate study through the DBT-JRF program. DP acknowledges Swarnajayanti fellowship (DST/SJF/LSA-02/2015-2016) from DST. DP and PKV thank the Department of Biotechnology, inStem for core funding.

Competing interests

The authors declare no competing or financial interests.

References

Lei, K., Mckinney, S. A., Ross, E. J., Lee, H. C. and Alvarado, A. S., (2019). Cultured pluripotent planarian stem cells retain potency and express proteins from exogenously introduced mRNAs. BioRxiv 573725.
Hayashi, T., Asami, M., Higuchi, S., Shibata, N. and Agata, K. (2006). Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting. Dev Growth Differ 48(6): 371-380.
Higuchi, S., Hayashi, T., Hori, I., Shibata, N., Sakamoto, H. and Agata, K. (2007). Characterization and categorization of fluorescence activated cell sorted planarian stem cells by ultrastructural analysis. Dev Growth Differ 49(7): 571-581.
Mohamed Haroon, M., Lakshmanan, V., Sarkar, S. R., Lei, K., Vemula, P. K. and Palakodeti, D. (2021). Mitochondrial state determines functionally divergent stem cell population in planaria. Stem Cell Reports 16(5): 1302-1316.
Niu, K., Xu, H., Xiong, Y. Z., Zhao, Y., Gao, C., Seidel, C. W., Pan, X., Ying, Y. and Lei, K. (2021). Canonical and early lineage-specific stem cell types identified in planarian SirNeoblasts. Cell Regen 10(1): 15.
Wagner, D. E., Wang, I. E. and Reddien, P. W. (2011). Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration. Science 332(6031): 811-816.
Wang, I. E., Wagner, D. E. and Reddien, P. W. (2018). Chapter 20: Clonal analysis of planarian stem cells by subtotal irradiation and single-cell transplantation. Methods Mol Biol 1774: 475-495.

简介

[摘要] 涡虫是自由生活的扁虫，它们成为了解再生和干细胞生物学的关键模型系统。通过荧光激活细胞分选 (FACS) 纯化新生细胞（涡虫的成体干细胞群）的能力极大地增加了我们对多能性、专业化和异质性的理解。迄今为止，基于 FACS 的新生细胞纯化方法依赖于区分增殖细胞 (>2N) 的核染料，因为新生细胞是唯一分裂的体细胞。然而，这种方法不能区分新生细胞群内的功能状态。我们的工作表明，在新生细胞中，多能干细胞 (PSC) 与低线粒体含量相关，这一特性可用于纯化富含 PSC 的群体。使用线粒体染料 MitoTracker Green (MTG) 和核染料 SiR-DNA，我们描述了一种分离 PSC 的方法，该方法可行且与下游实验（如移植和细胞培养）兼容。在本协议中，我们详细描述了涡虫中新生细胞分离的样品制备和 FACS 门控。

[背景] 对活细胞中的细胞核进行染色的荧光染料用于从涡虫中增殖干细胞的荧光激活细胞分选 (FACS)。在一项开创性的工作中，Hayashi等人。使用 Hoechst 对涡虫细胞进行染色并将细胞分为三个群体，称为 X1、X2 和 Xins 群体（图 2）和（Hayashi等人，2006）。这允许在涡虫研究领域中对增殖细胞（X1 细胞）进行分类（Hayashi等人，2006）。后来，Hoechst 染色剂被发现有毒，因此，对于需要活细胞的下游应用，必须使用基于前向散射 (FSC) 的不同 FACS 门 X1(fs) (Wagner et al ., 2011) 。该群体是通过在 FSC 与 SSC 图中对 X1 群体进行反向门控获得的（Wang等人，2018 年）。然而，X1(fs) 被分化细胞严重污染。随后，提出了核染料 SiR-DNA 作为 Hoechst 的替代品，用于获得具有高活力的增殖干细胞（Lei等人，2019；Niu等人，2021）。然而，这种使用核染料的流式细胞术方法无法区分多能干细胞群和特化干细胞群。
可以区分多能干细胞和特化干细胞的方法可以促进我们对涡虫干细胞生物学和功能的理解。由于新生细胞的细胞质不足和细胞器减少，我们推断细胞器的复杂性可能是区分不同细胞状态的方法（Higuchi等，2007）。为此，我们使用了线粒体，一种在新陈代谢和信号传导中起重要作用的细胞器。对哺乳动物干细胞的研究表明，线粒体状态，包括线粒体活性、含量、形态和数量在不同的细胞状态之间存在差异。我们最近报道了将 MitoTracker Green (MTG) 染色与核染料（如 Hoechst 和 SiR-DNA）结合用于纯化多能干细胞（Mohamed Haroon等人，2021）。我们的数据表明，与高 MTG 细胞相比，具有低 MTG 的增殖细胞群富含多能细胞，如更高的移植效率所示（MTG 低细胞约为 65%，MTG 高细胞约为 30%）。此外，该方法可用于从 X2 群体中富集 2N 干细胞，X2 群体是干细胞和有丝分裂祖细胞的混合物。为此，基于 MTG 和 FSC 将 X2 门分为四个群体，这表明了大小。我们发现 2N PSC 在低 MTG 和高 FSC 门中富集（Mohamed Haroon等人，2021）。相比之下，具有低 MTG 和低 FSC 的 X2 细胞主要是早期有丝分裂后祖细胞。观察到 X2 高 MTG 细胞，无论其大小如何，都是晚期祖细胞（Mohamed Haroon等人，2021）。在这里，我们为各种 MTG 种群的涡虫分离、染色和 FACS 门控提供了一个分步协议。

关键字：涡虫, 再生, 干细胞, 多能性, FACS, 线粒体

材料和试剂

1. 50 mL离心管（Tarsons，目录号： 546021）

2. 15 mL离心管（Tarsons，目录号： 546041）

3. 35 毫米培养皿

4. 60 毫米培养皿

5. Falcon 5 mL 圆底 FACS 管（BD，Falcon，目录号： 352054）

6. Sphero ^TMRainbow 荧光颗粒（BD Biosciences，目录号： 556291）

7. BD FACS Accudrop 珠子（BD，目录号： 345249）

8. 0.45 μ m Millex-HV 注射器过滤器单元（Merck Millipore，目录号： SLHVR33RS）

9. 0.22μm Millex-GV注射器过滤器单元（Merck Millipore，目录号： SLGVR33RS ）

10. 40 µm 细胞过滤器（BD Falcon，目录号： 352340）

11. 23 号手术刀片（李斯特）

12. 牛血清白蛋白（Sigma-Aldrich，目录号： A2153）

13. Hoechst 33342（Invitrogen，目录号： H3570）

14. MitoTracker Green FM（Invitrogen，目录号： M7514）

15. SiR-DNA（Cytoskeleton, Inc.目录号： CY-SC007）

16. DAPI（Sigma-Aldrich，目录号： D9542）

17. 碘化丙啶（Sigma-Aldrich，目录号： P4864 ）

18. MEM必需氨基酸溶液（50 × ）（ThermoFisher Scientific，Gibco ^TM，目录号： 11130051）

19. MEM非必需氨基酸溶液（100 × ）（ThermoFisher Scientific，Gibco ^TM，目录号： 11140050）

20. MEM维生素溶液（100 × ）（ThermoFisher Scientific，Gibco ^TM，目录号： 11120052）

21. 丙酮酸钠（100 × ）（ThermoFisher Scientific，Gibco ^TM，目录号： 11360070）

22. 100 ×青霉素链霉素（ThermoFisher Scientific，Gibco ^TM，目录号： 15070063）

23. L-谷氨酰胺（ThermoFisher Scientific，Gibco ^TM，目录号： 25030081）

24. 胎牛血清（Himedia，目录号： RM9955）

25. 氯化钠

26. CaC ₃

27. 硫酸镁₄

28. 氯化镁₂

29. 氯化钾

30. 碳酸氢钠₃

31. NaH ₂PO ₄

32. 氯化钾

33. 葡萄糖

34. HEPES（游离酸）

35. HEPES（钠盐）

36. 氯化锰₂

37. KH ₂PO ₄

38. D-生物素

39. D-葡萄糖

40. D-海藻糖

41. 曲辛

42. Montjuïc 盐（见食谱）

43. 含 BSA (CMFB) 的无钙、镁缓冲液（参见食谱）

44. IPM + 10% FBS（见食谱）

设备

1. 冷冻离心机（Eppendorf，型号：5810R）

2. FACS 分拣机（BD ARIA III 或 BD ARIA Fusion）

软件

1. FlowJo

2. BD FACS DIVA

程序

A. 细胞悬液的制备

1. 将饥饿至少 7 天的所需数量的蠕虫分开，并用新鲜的 1 × Montjuïc 盐清洗。

注意：此协议针对涡虫物种 Schmidtea mediterranea 进行了优化，并且可以使用有性和无性生物型。 所需的蠕虫数量取决于下游应用所需的细胞数量。根据我们的经验，约 0.7 厘米大小的 35 条蠕虫将产生约 100,000 至 150,000 个 X1 细胞。

2. 将蠕虫转移到 60 毫米或 35 毫米培养皿的盖子上。完全去除 1 × Montjuïc 盐。

3. 立即将 ±100 μL的 CMFB 添加到涡虫中（图 1A）。

注意：CMFB 的体积是近似的，因为它应该足以覆盖所有涡虫。体积可以根据具体要求放大或缩小。

4. 使用手术刀, 将蠕虫切成尽可能小的碎片 (图 1B)。用纸巾间歇性地擦拭手术刀，以去除粘液。确保没有大碎片残留。

5. 将组织切成小块后，加入 1 mL 的 CMF B。

6. 使用无菌手术刀切割其末端，制作宽口径 1 mL 移液器尖端（图 1C）。使用这种大口径移液器，将组织碎片转移到新的 50 mL 离心管中。

注意：组织碎片可能会粘在表面上。如果是这样，用多余的 CMFB 冲洗组织并将其转移到离心管中。在我们的实验室中，我们将组织碎片放入约 5 mL 的 CMFB 中。

7. 通过轻轻吹打多次，使用 1 mL 移液器机械分离细胞。移液约 20-30 次后，让离心管静置约 2-3 分钟。未解离的组织碎片会沉淀下来。将含有细胞的上清液转移到新的 50 mL 管中，而不会干扰沉淀的组织碎片。留下一些上清液，这样未解离的片段就不会与上清液一起出现（图 1D）。

8. 将另外 3-4 mL 的 CMFB 溶液添加到未分离的碎片中，并通过轻轻移液再次分离。

9. 重复步骤A7和A8 ，直到所有组织碎片完全解离。

注意：步骤A 7 和A 8 是为了尽可能减少细胞死亡，因为已经解离的细胞的移液是不必要的，并且可能会导致细胞死亡。

10. 收集所有细胞悬浮液并通过 40 μm过滤器将其过滤到新鲜的 50 mL 离心管中。

11. × g在 4 °C 下将应变细胞悬浮液离心10 分钟。在此阶段观察到暗细胞颗粒（图 1E）。

12. 丢弃上清液并将细胞颗粒重新悬浮在 IPM + 10% FBS 培养基中。媒体的体积取决于使用的蠕虫的数量和大小。对于约 0.7 厘米的约 40 条蠕虫，可以使用 10 mL 的介质。

图 1.细胞悬液的制备。

A. 将涡虫放入 35 mm 培养皿的盖子中，并添加约 100 μL CMFB 。 B. 切割涡虫碎片的代表性图像。 C. 与普通移液器（左）相比，带有切割尖端的 1 mL 移液器的代表性图像（右）。 D. 通过移取细胞悬浮液约 20-30 次进行机械破碎后，让溶液沉淀下来（左离心管），将上清液转移到新的离心管（右）中，将新鲜的 CMFB 添加到未解离的片段中.有关详细信息，请参阅步骤 A7-A9 。 E. 细胞过滤和离心后的细胞沉淀。

B. 细胞悬液染色

1. 加入 40 μ g/mL Hoechst 33342，室温避光孵育 40 分钟。以约 10 分钟的间隔间歇地混合溶液。

2. 40 分钟后，将 100 nM MTG 添加到相同的细胞悬液中，并在室温下在黑暗中再孵育 20 分钟。

3. 与 MTG 孵育后，将细胞悬液在 4 °C 下在摆动桶转子中以 290 × g离心10 分钟。丢弃上清液并将细胞颗粒重新悬浮在 IPM + 10% FBS 培养基中。

最终体积可以根据分拣机的效率进行调整。在我们的实验室中，对于约 0.7 厘米的约 40 条蠕虫，我们使用 3 mL 的 IPM + 10% FBS 介质。

4. 最后，在细胞悬浮液中加入 1 μg/mL 碘化丙啶，以区分活/死细胞。

5. 对于用于移植或体外培养实验的细胞的 FACS 分选，在步骤 B1 应使用SiR-DNA (1 μ M) 代替 Hoechst 33342。在步骤 B4，对于活/死细胞的区分，使用 10 μ g/mL 的 DAPI 代替碘化丙啶。染色协议的其余部分保持不变。

C. X1 和 X2 MTG 群体的 FACS 门控

1. 对于排序，在 20 psi 护套压力下使用 100 μ m 尖端。使用专有的 BD 鞘液。

注意：在每次分选之前，我们都会通过使用 Sphero ^TMRainbow 荧光颗粒设置适当的激光延迟和使用 BD FACS Accudrop 珠子设置适当的激光延迟来定期校准仪器。此步骤应根据所使用的仪器来完成，并且信息通常随仪器手册提供。

2. 在 FACS 机器中运行 Hoechst 33342 和 MTG 染色细胞。调整前向散射 (FSC) 和侧向散射 (SSC) 电压并制作门 (P1) 以排除碎片和死细胞。

3. 接下来，使用 FSC A（面积）与 FSC W（宽度）点图 (P2) 对单峰进行门控。

4. 在此单线态种群中，选择指示为碘化丙啶阴性事件 (P3) 的活细胞。

5. (Mohamed Haroon et al. , 2021)所示，设置 X1、X2 和 Xins 种群的大门ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.stemcr.2021.03.022","ISSN":"22136711","PMID":"33861990","abstract":"Mitochondrial state changes were shown to be critical for stem cell function. However, variation in the mitochondrial content in stem cells and the implication, if any, on differentiation is poorly understood. Here, using cellular and molecular studies, we show that the planarian pluripotent stem cells (PSCs) have low mitochondrial mass compared with their progenitors. Transplantation experiments provided functional validation that neoblasts with low mitochondrial mass are the true PSCs. Further, the mitochondrial mass correlated with OxPhos and inhibiting the transition to OxPhos dependent metabolism in cultured cells resulted in higher PSCs. In summary, we show that low mitochondrial mass is a hallmark of PSCs in planaria and provide a mechanism to isolate live, functionally active, PSCs from different cell cycle stages (G0/G1 and S, G2/M). Our study demonstrates that the change in mitochondrial metabolism, a feature of PSCs is conserved in planaria and highlights its role in organismal regeneration.","author":[{"dropping-particle":"","family":"Mohamed Haroon","given":"Mohamed","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lakshmanan","given":"Vairavan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sarkar","given":"Souradeep R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lei","given":"Kai","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Vemula","given":"Praveen Kumar","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Palakodeti","given":"Dasaradhi","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Stem Cell Reports","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2021","5","11"]]},"page":"1302-1316","publisher":"Elsevier","title":"Mitochondrial state determines functionally divergent stem cell population in planaria","type":"article-journal","volume":"16"},"uris":["http://www.mendeley.com/documents/?uuid=4996400a-7268-3b19-8b46-9fec9e2a3ab9"]}],"mendeley":{"formattedCitation":"(Mohamed Haroon et al., 2021)","plainTextFormattedCitation":"(Mohamed Haroon et al., 2021)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}。

图 2 。 X1、X2 和 Xins 种群的代表性 FACS 门控。

有关详细信息，请参阅步骤 C2-C5。 FSC-前向散射，SSC-侧向散射，FSC-W宽度，FSC-A面积。 Hoechst 33342 的蓝色（Hoechst Blue）发射可以使用 450/20 带通滤光片收集，红色发射（Hoechst 红）使用 630/40 带通滤光片。

6. 要获得具有富集多能细胞的群体，请在 X1 内门控 MTG Low 细胞。类似地，丰富 X1 细胞内与 MTG 高对应的谱系启动人口门（图 3A）。

7. X2 门也可以分为 MTG Low 和 MTG High 单元。为了丰富 X2 中的 2N 干细胞，根据 MTG 和 FSC 参数制作四个门（图 3B）。

注意：C6-C7：根据目前的理解，在排除 X1 和 X2 群体中的分化后代时，不可能标记准确代表干细胞的 MTG Low 细胞的确切荧光强度或百分比。因此，为了保持一致性，可以将总体分为等百分比的 MTG Low 和 MTG High。

图 3。 (A) X1 和 (B) X2 细胞内的 MitoTracker Green (MTG) 种群的门控策略。

为保持一致性，应对等百分比的 MTG 低和高人群进行门控。 HFSC-高 FSC，LFSC-低 FSC。

D. 用于 SiR-DNA 2N 和 4N MTG 群体的 FACS 门控

1. 如上所述运行 SiR-DNA 染色细胞并设置门 P1 和 P2，以消除碎片和双峰。接下来，门 DAPI 负细胞 (P3) 以获得活细胞。

2. 在活细胞内，门 SiR-DNA 2N (P4) 和 4N (P5) 种群（图 4）。

注意：与 Hoechst 染色不同，SiR-DNA 染色在 FACS 图中不能很好地分离 2N 和 4N 细胞。因此，为确保准确的门控，请使用图 4 中的参考图和其他 Sir-DNA 图（Lei 等人，2019）。或者，可以使用 6000 拉德辐照动物（辐照动物后 2 天以上）作为没有增殖干细胞的对照（Wagner等人，2011）。

图 4 。 使用 SiR-DNA 核染料对 2N（P4 门）和 4N（P5 门）细胞进行代表性 FACS 门控。有关详细信息，请参阅步骤 D1-D2。

3. 要获得与 X1 等效的总体，首先通过在 FSC 与 SSC 图中反向门控 X1 细胞来确定 X1(fs) 门（图 5）（Wagner等人，2011）。在 X1(fs) 种群中，门控 SiR-DNA 4N 种群，如前面的步骤 D2 中确定的（图 5）。

4. 同样，通过在 FSC 与 SSC 图中反向门控 X2 细胞来确定 X2(fs) 门，并在 X2(fs) 门内对 SiR-DNA 2N 种群进行门控（图 5）。

5. 设置 MTG 低和高的门，以及 4N 和 2N 门内的 FSC 低和高，类似于步骤 C7-C8 中解释的赫斯特染色细胞。

图 5。 用于在 X2(fs) 和 X1(fs) 门内分类 2N 和 4N SiR-DNA 种群的代表性 FACS 图。

执行此门控是为了减少 X1 细胞和 X2 细胞的交叉污染，因为与 Hoechst 不同，SiR-DNA 染料不能很好地解决细胞周期阶段。

E. FACS 分拣

1. 对含有 2 mL IPM + 10% FBS 介质的 5 mL FACS 管中的细胞进行排序。使用适当的适配器将收集室保持在 4 °C 。

注意：我们通常使用 BD FACS Aria III 和 BD FACS Aria Fusion 分拣机中描述的“纯度”模式进行分拣。此类分选精度模式因仪器而异，用户应参考仪器手册进行相应设置。用户可以在 FACS 机器中运行分选的样品，以确保分选的纯度。此外，可以对新生细胞标记物（如 piwi-1）进行荧光原位杂交或免疫染色，以检查分选样品的纯度。

2. 使用摆动桶转子在 4 °C 下以 300 × g将分选的细胞离心10 分钟。丢弃上清液并根据下游应用将细胞重新悬浮在适当的体积中。

注意：分选约 100,000 至 150,000 个细胞后的细胞沉淀会非常小，可能不容易看到。

数据分析

使用 BD FACS DIVA 软件分析流式细胞术图。也可以使用 FlowJo 等替代软件。分离的细胞群通过至少三个独立重复的 PIWI-1 标记表达和移植实验来表征（Mohamed Haroon等人，2021）。使用未配对的双尾学生t检验进行统计分析。

食谱

1. 1 × Montjuïc 盐

氯化钠 1.6 毫米

CaC ₃1 毫米

硫酸镁₄1 毫米

氯化镁₂0.1 毫米

氯化钾 0.1 毫米

碳酸氢钠₃1.2 毫米

酸碱度 7.0

2. 含 BSA (CMFB) 的无钙、镁缓冲液（适用于 1,000 mL）

NaH ₂PO ₄ 400 毫克

氯化钠 800 毫克

氯化钾 1,200 毫克

碳酸氢钠₃ 800 毫克

注：上述盐的 10倍浓度可单独制备（过滤和高压灭菌）并作为储备储存。

葡萄糖 240 毫克

HEPES 15 毫米

酸碱度 7.3

牛血清白蛋白 1%

注意：应将 1% BSA 新鲜添加到缓冲液中。

3. IPM + 10 % FBS（对于 1 , 000 mL）

HEPES（游离酸） 7 , 208.8 毫克

HEPES（钠盐） 3，514.1毫克

氯化钠 985.4 毫克

碳酸氢钠₃ 800.1 毫克

氯化钾 26.4 毫克

氯化钙₂ 113.23 毫克

硫酸镁₄ 44.02 毫克

MnCl ₂ 0.19 毫克

KH ₂PO ₄ 68.5 毫克

D-生物素 0.3 毫克

D-葡萄糖 300 毫克

D-海藻糖 50 毫克

三辛 2.5 毫克

MEM 必需氨基酸 2 毫升

MEM 非必需氨基酸 5 mL

MEM 维生素溶液 3 毫升

丙酮酸钠 14 毫升

青霉素/链霉素 10 mL

L-谷氨酰胺 2 毫升

胎牛血清 100 毫升

酸碱度 7.3

致谢

该协议主要改编自我们实验室最近发表的工作（Mohamed Haroon等人，2021）。作者感谢中央成像和流式细胞仪设施 (CIFF-BLiSc 校园) 提供最先进的流式细胞仪设施。 MMH 感谢 DBT 通过 DBT-JRF 计划资助研究生学习。 DP 承认来自 DST 的 Swarnajayanti 奖学金 (DST/SJF/LSA-02/2015-2016)。 DP 和 PKV 感谢 inStem 生物技术部的核心资助。

利益争夺

作者声明没有竞争或经济利益。

参考

1. Lei, K., Mckinney, SA, Ross, EJ, Lee, HC 和 Alvarado, AS, (2019)。培养的多能涡虫干细胞保留效力并从外源引入的 mRNA 中表达蛋白质。 BioRxiv 573725 。

2. Hayashi, T.、Asami, M.、Higuchi, S.、Shibata, N. 和 Agata, K. (2006)。通过荧光激活细胞分选分离涡虫 X 射线敏感干细胞。开发增长差异48(6): 371-380。

3. Higuchi, S.、Hayashi, T.、Hori, I.、Shibata, N.、Sakamoto, H. 和 Agata, K. (2007)。通过超微结构分析对荧光激活细胞分选的涡虫干细胞进行表征和分类。开发增长差异49(7): 571-581。

4. Mohamed Haroon, M.、Lakshmanan, V.、Sarkar, SR、Lei, K.、Vemula, PK 和 Palakodeti, D. (2021)。线粒体状态决定了涡虫中功能不同的干细胞群。干细胞报告16(5): 1302-1316。

5. Niu, K., Xu, H., Xiong, YZ, Zhao, Y., Gao, C., Seidel, CW, Pan, X., Ying, Y. 和 Lei, K. (2021)。在涡虫 SirNeoblasts 中鉴定的典型和早期谱系特异性干细胞类型。细胞再生10（1）：15。

6. Wagner, DE, Wang, IE 和 Reddien, PW (2011)。克隆新生细胞是多能成体干细胞，是涡虫再生的基础。科学332（6031）：811-816。

7. Wang, IE, Wagner, DE 和 Reddien, PW (2018)。第 20 章：通过次全照射和单细胞移植对涡虫干细胞进行克隆分析。 方法 Mol Biol 1774：475-495。

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引用：Mohamed Haroon, M., Vemula, P. K. and Palakodeti, D. (2022). Flow Cytometry Analysis of Planarian Stem Cells Using DNA and Mitochondrial Dyes . Bio-protocol 12(2): e4299. DOI: 10.21769/BioProtoc.4299.