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May 2019

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Strategy of Isolating ‘Primed’ Tumor Initiating Cells Based on Mitochondrial Transmembrane Potential
基于线粒体跨膜电位分离“启动”肿瘤起始细胞的策略   

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Abstract

Various stem cells have been found to be dependent on mitochondrial energetics. The role of mitochondria in regulating the self-renewal of normal stem cells and stem-like tumor initiating cells (TICs) is increasingly being appreciated. We proposed that TIC populations have a sub population of cells that are “primed” by mitochondria for self-renewal. Using ovarian cancer model, we have developed a protocol to identify and isolate these “primed” cells using Fluorescence-Assisted Cell Sorting (FACS). We combined live cell stains for a functional marker of TICs and for mitochondrial transmembrane potential to enrich TICs with higher mitochondrial potential that form in vitro spheroids 10-fold more than the other TICs with lower mitochondrial potential. This protocol can be directly used or modified to be used in various cell types. Thus, this protocol is anticipated to be invaluable for the basic understanding of mitochondrial and energetic heterogeneity within stem cell population, and may also prove valuable in translational studies in regenerative medicine and cancer biology.

Keywords: Tumor initiating cells (肿瘤启动细胞), Mitochondrial energetics (线粒体能量), Stem Cell Priming (干细胞启动), Self-Renewal (自我更新), FACS (荧光激活细胞分选术)

Background

Cellular heterogeneity in tumors poses tough challenges for cancer therapy (Magee et al., 2012). Certain tumors are arranged hierarchically with the chemotherapy resistant tumor initiating cells (also called cancer stem cells) residing at the bottom of the cellular hierarchy as they maintain the tumor through self-renewal and differentiation (Magee et al., 2012). Plasticity of the tumor initiating cells (TICs) makes them difficult to identify, isolate and study. Heterogeneity and plasticity of energetics of TICs is only beginning to be appreciated and is far from clear (Martinez-Outschoorn et al., 2017) (Intlekofer and Finley, 2019). Moreover, energetics status of TICs appears to be tumor specific, and certain TICs are dependent on mitochondrial energetics and can be eliminated by mitochondrial inhibitors (Pasto et al., 2014; Sancho et al., 2015; Viale et al., 2015; Martinez-Outschoorn et al., 2017; Spurlock et al., 2019).


Mitochondrial energetics is maintained by complex biochemical regulation of multiple bioenergetic parameters, including mitochondrial transmembrane potential maintained across the mitochondrial inner membrane (ΔΨ), which is consumed to generate ATP, heat, or reactive oxygen species. Higher mitochondrial transmembrane potential is expected to maintain higher ATP synthesis depending on various other parameters like substrate availability, etc. Previously, stem cells isolated based on mitochondrial transmembrane potential have been found to differ in their stem cell properties (Schieke et al., 2008; Sukumar et al., 2016). Recently, we developed a flowcytometric protocol to simultaneously sort cells for ΔΨ, using the red fluorescent potentiometric dye TMRE, and the activity of a functional TIC marker aldehyde dehydrogenase (ALDH), using the AldeFluor reagent (Figures 1-2) (Spurlock et al., 2019). Using such a flow cytometric sorting strategy, we were able to identify a subpopulation of human ovarian TICs with 10 fold higher self-renewing and proliferating capacity, as determined by in vitro limiting dilution assay. Thus, our protocol enables teasing out mitochondrial heterogeneity in stem cell populations.


The role of mitochondrial energetics in maintenance of normal stem cells and TICs is more appreciated as our understanding of stem cell energetics increases (Viale et al., 2015; Chandel et al., 2016; Intlekofer and Finley, 2019). Adult stem cells are majorly quiescent and considered to be highly glycolytic and release from quiescence involves metabolic/energetic shifts that remain poorly understood (Margineantu et al., 2002; Ito and Suda, 2014; Folmes and Terzic, 2016). We proposed that a subpopulation of ovarian TICs with particularly elevated mitochondrial transmembrane potential, associated with other specific mitochondrial properties, are primed for greater self-renewal and proliferation ability (Spurlock et al., 2019). Similar mitochondria based priming has been observed in hematopoietic lineage and has been linked to exit from quiescence and entry into the cell cycle for self-renewal and differentiation (Liang et al., 2020). Our protocol specifically isolates these mitochondria primed stem cells (mpSCs) by combining a TIC marker (Aldh activity) with ΔΨ. Aldh activity is a TIC marker for several tumor types, including ovarian, breast, cervical, lung, liver, prostate, bone marrow, skin, colon, and pancreatic (Marcato et al., 2011; Toledo-Guzmán, et al., 2019; Vassalli, 2019). We developed this protocol in human ovarian cancer cell lines; however, we have also performed these assays in human ovarian cancer cells derived from a mouse xenograft model as well as patient ascites. The sorted cells can be studied for stem cell or other properties with relevant methods. We have studied mitochondrial structural and functional properties of the sorted cells using our novel assay mito-SinCe2 (Spurlock et al., 2019) (example in Figure 3). This protocol is a simple and valuable tool for the isolation and study of normal and neoplastic stem cells from cell lines as well as primary cell populations. Thus, this protocol will allow a basic understanding of mitochondrial and energetic heterogeneity within stem cell population, and may also prove valuable in translational studies in regenerative medicine and cancer biology.


Materials and Reagents

  1. 1.5 ml microfuge tubes (Eppendorf, catalog number: 022363204)

  2. 15 ml centrifuge tubes (Corning, catalog number: 430790)

  3. 5 ml flow cytometry tubes (Falcon, catalog number: 352003)

  4. 96-well Ultra-Low Attachment Plates (Corning, catalog number: 3474)

  5. 8-well Lab-Tek II Chambered Coverglass (Nunc, catalog number: 155409)

  6. Appropriate cell culture materials and reagents for cells in use

  7. DPBS (Sigma, catalog number: 59331C-1000ML)

  8. Trypsin 0.05% (GE Healthcare Life Sciences, catalog number: SH30236.02 or similar)

  9. AldeFluor Kit (StemCell Technologies, catalog number: 01700)

    For lineages where ALDH activity does not mark stemness, our protocol can be modified for use with any lineage by including lineage specific culture conditions and live cell markers (CD133 etc.), ensuring minimal or no overlap with TMRE fluorescence.

  10. Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) (Invitrogen, catalog number: T669)

  11. RPMI medium for the A2780 ovarian cancer cell line in use (Corning, catalog number: 15-040-CM) or appropriate base media

  12. N1 supplement (Sigma, catalog number: N6530-5ML)

  13. Insulin (Sigma, catalog number: 10516-5ML)

  14. Sodium pyruvate (GE Healthcare Life Sciences, catalog number: SH30239.01)

  15. L-glutamine (GE Healthcare Life Sciences, catalog number: SH30034.01)

  16. Penicillin-Streptomycin Solution (GE Healthcare Life Sciences, catalog number: SV30010)

  17. FGF (Sigma, catalog number: F0291-25ug)

  18. EGF (Sigma, catalog number: E9644-0.2ug)

  19. TIC media (see Recipes)

Equipment

  1. BD FACSAria II or similar with cytometer setup and tracking (CS&T) beads, accudrop calibration beads, 100 μm FACS cell nozzle and a minimum of 488 nm and 561 nm laser and with emission filters of 525/50 BP for AldeFlour and 576/26 BP for TMRE

  2. Centrifuges for 1.5 ml microfuge tubes and 15 ml tubes

  3. Standard biosafety hood with laminar flow

  4. Standard incubator for cell culture maintained at 37 °C and 5% CO2 with humidification

  5. Light microscope with 4× and 10× objectives

  6. Any confocal microscope with 555 nm laser (e.g., Zeiss LSM700)

Software

  1. BD FACSDiva (Version 8.0.1) (with automatic compensation calculation feature) or similar for flow cytometric sorting and analyses

Procedure

See Figure 1.

Note: Given the protocol pertains to live cells, carefully consider the time taken for each procedure for maximizing the rigor and reproducibility of the procedure. Do not halt the protocol at any step and implement efficient planning for minimizing time taken for each procedure. The time taken for each procedure in our hands is as follows:

  1. Sample preparation for FACS: 2 h

  2. FACS to isolate four populations: 2 h per sample tube.

  3. Spheroid formation and ELDA: 15 d for the cell lineage used here. This time may vary with cell lineage.


    Figure 1. Graphical representation of work flow. Prepare control and sample tubes by bringing cells to single cell suspension and staining as described. Use control tubes to set parameters on the sorter and then sort four populations. The double positive population is enriched for mpSCs.


  1. Sample preparation for Fluorescence-Activated Cell Sorting (FACS)

    1. Preparations for Staining:

      1. Reconstitute, aliquot and store reagents of the AldeFluor kit and TMRE according to manufacturer guidelines. Check manufacturer protocol for chemistry of the AldeFluor reagent.

      2. Culture cells normally until ready to begin protocol. Two days prior to sorting, seed cells as typical to ~40% confluence in normal growth media, ensuring sorted populations will have enough cells required for subsequent experiments as determined from a mock experiment with 10 million cells.

        Note: Although we developed the protocol with ovarian cancer cells, it can be used for any other cells with appropriate culture conditions and stem cell medium (in subsequent steps). Moreover, the staining and sorting protocol is also amenable to be used for cells directly isolated from any tissue or tumor xenografts. The sorted cells can be injected directly to animals for analyses of stem cell properties employing in vivo limiting dilution assay or other specific assays.

      3. Warm aliquots of trypsin, DPBS, culture media, and AldeFluor buffer (from the AldeFluor kit) to 37 °C at least three hours prior to staining.

      4. Trypsinize cells and transfer them to a 15 ml conical tube. Deactivate trypsin by adding an equal amount of culture media to the cell suspension. Make a homogenous cell suspension by pipetting thoroughly to ensure single cell suspension.

        Note: As written, this protocol is for cell lines. To modify for primary cells and tissues, bring cells to a homogenous single cell suspension using appropriate techniques and begin from Step A1e.

      5. Count cells from the homogenous cell suspension (in Step A1d) using preferred method.

      6. Wash cells by first centrifuging cell suspension at 300 × g for 10 min at Room Temperature (RT), and removing the supernatant and resuspending cells in 3 ml warm DPBS. Finally, centrifuge cells at 300 × g for 10 min at RT.

    2. Staining with AldeFluor and TMRE:

      1. Aspirate the supernatant from the cell pellet (obtained in Step A1f) and resuspend cells in warm AldeFluor buffer to the concentration of 2 × 106 cells/ml, pipetting thoroughly to ensure single cell suspension.

      2. Label 3, 1.5 ml microfuge tubes as “Unstained”, “AldeFluor”, and “TMRE” to serve as compensation controls to correct for overlap in the emission spectra of the AldeFluor reagent and TMRE. Label a fourth 1.5 ml microfuge tube as “DEAB” that, along with the “TMRE” tube, will serve as biological and gating controls for the respective AldeFluor and TMRE staining. Finally, label one 15 ml conical tube as “AldeFluor+TMRE” for the experimental sample to be sorted.

      3. Distribute 1 × 106 cells (0.5 ml) each from cells suspension in Step A2a in Unstained, AldeFluor, TMRE and DEAB tubes. Transfer the rest of the cells to the AldeFluor+TMRE tube.

      4. Follow manufacturer’s protocol to stain cells with AldeFlour reagent:

        1. Add 5 μl reagent per 2 × 106 cells in the AldeFluor, DEAB and AldeFluor+TMRE tubes.
        2. Inhibit Aldh activity in the DEAB tube using 5 μl per 1 × 106 cells.
      5. Incubate all five tubes at 37 °C and 5% CO2 for 1 h (or as optimized from manufacturer’s protocol). Tap tubes to resuspend cells every 15 min.

      6. Centrifuge cells in all 5 tubes at 300 × g for 10 min at RT.

      7. While the previous step is ongoing, prepare 25 nM solution of TMRE in warm AldeFluor buffer.

      8. Stain cells in the TMRE and AldeFluor+TMRE tubes with TMRE by aspirating the supernatant and resuspending in the 25 nM TMRE solution to 2 × 106 cells/ml.

      9. Resuspend cells in the Unstained, AldeFluor, and DEAB tubes in warm AldeFluor Buffer to 2 × 106 cells/ml.

      10. Incubate tubes at 37 °C and 5% CO2 for 15 min.

      11. Centrifuge all five tubes at 300 × g for 10 min at RT.

      12. Label five 5 ml flow cytometry tubes as in Step A2b.

      13. Transfer appropriately stained cells to equivalently labeled flow cytometry tubes by aspirating the supernatant and resuspending cells in AldeFluor buffer to a concentration of 5 × 106 cells/ml. Ensure no bubbles are introduced to the tubes.

      14. This TMRE staining protocol effectively stains energetically active mitochondria in any given cell. However, TMRE staining of mitochondria should be confirmed by adding stained cells to a live cell chambered coverglass, allowing them to settle for 5 min, and looking at them under a confocal microscope (Figure 2A). Staining with MitoTracker Green may be necessary to ensure the staining is mitochondrial. Protocol described in (Mitra and Lippincott-Schwartz, 2010). If TMRE does not incorporate to mitochondria, TMRE staining should be re-optimized by varying first TMRE concentration and incubation time in a given cell density.


  2. Flow cytometry data acquisitions for sorting (as optimized using the BD FacsAria II)

    Note: The following precautions are necessary:

    1. Data acquisition and sorting should be accomplished within 2 h or less of transferring sample to 5 ml flow cytometry tubes. Longer time taken may cause loss of signal. For large numbers of cells, seed multiple plates so that the first set of cells can be sorted within two hours. Process the next set while the first is sorting and so on.

    2. Optimal sorter performance should be ensured by running cytometer setup and tracking (CS&T) beads. Drop delay should be calculated using accudrop calibration beads.

    1. Data acquisition can be performed using software such as BD FACSDiva (Version 8.0.1) or similar on the sorter.

    2. Attach the 100 μm nozzle to sort at 20 psi pressure. Maintain 4 °C for both the sample and collection tubes through the sort. Select the following parameters: Forward Scatter (FSC in linear scale) and Side Scatter (SSC in linear scale), channels for AldeFluor (Ex: 488 nm, Em filter: 525/50 BP; log scale) and TMRE (Ex: 561 nm, Em filter: 576/26 BP, log scale).

      Adjust parameters using the compensation controls as follows:

      1. Using the unstained sample do the following adjustments:

        1. Adjust PMT voltages of FSC and SSC to display and distinguish the target population of interest from cell debris.

        2. Gate out the cell debris using the parameters FSC Area (FSC-A) and SSC Area (SSC-A) (Figure 2B).

        3. Gate out cell doublets using FSC-Height (FSC-H), FSC-A, SSC Width (SSC-W) and FSC-Height (SSC-H) in two subsequent gates (Figures 2C, 2D).

        4. Adjust PMT voltages of Unstained sample to have the auto fluorescence signal peak below the second decade of the log scale for both TMRE (Figure 2E, left) and AldeFluor (Figure 2E, right).

      2. Using the TMRE stained sample, adjust PMT voltage for TMRE detection to ensure the signal is within the dynamic range (Figure 2F).

      3. Using the AldeFluor stained sample, adjust PMT voltage for AldeFluor detection to ensure the signal is within the dynamic range (Figure 2G).

      4. Once the voltages are finalized, record data with 20,000 events from the Unstained, AldeFluor and TMRE tubes, and use the automatic compensation calculation feature to calculate the compensation of fluorescence signal and apply to the experiment.

        Note: Compensation and biological controls may not be necessary for every sort, after reproducibility of the sort has been established. However, re-introduce controls if the staining has been modified or if dealing with samples with large inherent variations like patient samples. If dealing with patient samples, extra steps have to be taken for gating out dead cells.


      Figure 2. FACS strategy for enriching and depleting mitochondria primed TICs . A. Micrographs of iPSCs stained with TMRE as examples of good (left) and bad (right) incorporation of the dye into mitochondria. Scale bars represent 5 µm. B. Contour plot showing gating of whole cells using FSC-A and SSC-A, respectively, for the epithelial ovarian cancer cell line A2780-CP. C. Contour plot showing first gating of single cells using FSC-H and FSC-A for A2780-CP. D. Contour plot showing second gating of single cells using SSC-H and SSC-W for A2780-CP. E. Histograms showing auto-fluorescence of A2780-CP cells using TMRE parameters (left) and AldeFluor parameters (right) set within second decades of fluorescence intensity. F. Contour plot showing TMRE staining of A2780-CP cells within the dynamic range of the instrument. G. Contour plot showing AldeFluor staining of A2780-CP cells within the dynamic range of the instrument. H. Contour plot showing AldeFluor staining after inhibiting Aldh activity using DEAB. This plot marks the boundary of AldeFluor cells. I. Contour plot showing final sort gating of four populations of A2780-CP: AldeFluor+ TMRE+, AldeFluor+ TMRE, AldeFluor TMRE+, AldeFluor TMRE.


  3. FACS to isolate four populations: AldeFluor+ TMRE+, AldeFluor+ TMRE, AldeFluor TMRE+, AldeFluor TMRE

    1. After executing Step B2c, use biological/gating controls to set the sorting gates as follows:

      1. Record 20,000 events using DEAB tube and TMRE tube.

      2. Set thresholds for AldeFluor+ (Figure 2H, red line) and TMRE+ (Figure 2F, red line).

      3. Record 20,000 events using the AldeFluor+TMRE stained sample.

      4. Draw sorting gates around distinct populations within each quartile as shown in Figure 1H (black rectangles) for sorting the following cell populations: AldeFluor+ TMRE+, AldeFluor+ TMRE, AldeFluor TMRE+, AldeFluor TMRE.

      Note: Gating out debris was sufficient to exclude dead cells in our cell lines, where viability after acquiring a single cell suspension typically exceeded 90%. Fresh tissues and cells of other lineages may require a viability stain to exclude dead cells. Stains must be compatible with TMRE fluorescence (e.g., gating live cells by DAPI exclusion).

    2. Set the flow cytometer to record 100,000 events from the gated pre-sort sample. Use the BD FACSDiva (Version 8.0.1) software to determine the percentage of cells in each population. The AldeFluor+TMRE+ population is enriched for mitochondria primed TICs.

    3. For collection of population to be sorted, add 0.5 ml of either sterile culture media or appropriate stem cell media to four 5 ml flow cytometry collection tubes and label the tubes “AldeFluor+ TMRE+”, “AldeFluor+ TMRE”, “AldeFluor TMRE+” and “AldeFluor TMRE”. Place the collection tubes in the position in the FACSAria corresponding to the sorting gate (Figure 2I) for the population labeled on the tube.

      Notes:

      1. The TIC medium or stem cell medium composition may differ between lineages. Therefore, use the appropriate stem cell medium for the spheroid assay.

      2. If the FACS instrument is in a different location than that of the sample preparation, transfer cells, collection tubes, extra flow cytometry tubes, and aliquots of AldeFluor Buffer and cell culture media at RT in a sterile container protected from exposure to light.

      3. Distribution of TMRE fluorescence in the example cells provided here is bimodal, lending easily to separate positive and negative populations. Should the distribution be not bimodal, take the bottom 10% of the normal distribution for the negative population and the top 10% for the positive.

    4. Use 100 μm nozzle to sort at 20 psi pressure. Maintain 4 °C for both the sample and collection tubes through the sort. Maintain the flow rate to have a threshold rate of 9,000 to 10,000 events per second. Vortex the sample at medium speed before loading to the sample port. Maintain sample agitation at 200 rpm through the sort.

    5. Allow the sort to continue until the targeted cell number is reached for each population, replacing collection tubes as needed.

    6. Maintain sorted cells at 4 °C until needed for subsequent experiments.

    7. Before seeding or harvesting sorted cells, transfer cells to labeled 15 ml conical tubes and centrifuge at 300 × g for 10 min at 4 °C.

    8. Aspirate supernatant and resuspend cells in DPBS or desired cell culture media. Centrifuge at 300 × g for 10 min at 4 °C and aspirate the supernatant.

    9. Use these cell pellets or cell suspension for further experiments, as appropriate.


  4. Extreme Limiting Dilution Analysis (ELDA) (Hu and Smyth, 2009) to quantitate TIC frequency from spheroids formed by the sorted population.

    Note: Although this can be optional, we recommend performing this assay when using the sorting protocol for the first time in any new lineage or cell type. This is because the sorted population primed for maximal self-renewal and proliferation, as quantified by this assay, may or may not be similar to the ovarian cancer system shown here. This in vitro assay can also be replaced by the in vivo limiting dilution assay using xenografts.

    1. Spheroids are formed as follows:

      1. Based on the number of cells sorted, resuspend each population in stem cell media to a concentration of 104 cells/ml (final volume of at least 3 ml).

      2. Serially dilute a portion of the above suspension to 103, 102 and 10 cells/ml.

        Note: Each dilution of 10 fold can be achieved by adding 300 μl of each successive cell suspension to 2.7 ml stem cell media in 15 ml conical tubes. We always seeded cells using serial dilution based on counting live cells by Trypan Blue exclusion after the wash steps. However, seeding can also be accomplished by sorting the desired number of cells directly into each well of the 96-well Ultra-Low Attachment plate.

      3. Distribute each cell suspension among at least 24 wells in a 96-well Ultra-Low Attachment plate (100 μl /well). The more total wells used for each cell concentration, the higher the statistical power of the assay. Leave plates in a cell culture incubator. In the example experiment in Figure 3, we used 4 plates, one for each population. In each plate, we seeded 24 wells with 1 cell/well, 24 wells with 10 cells/well, 24 wells with 100 cells/well, and 24 wells with 103 cells/well.

      4. Every second day, examine each well using the 10× objective of a light microscope for the formation of dense spheroids with tight edges (Figure 3A).

      5. Spike cells every second day with 10% of the initial volume of stem cell media until conspicuous tumorspheres with tight edges are detected in the positive control group (Figure 3A); in the example provided Aldh+ (A+) groups are positive control while Aldh- (A-) groups are negative control (Figure 3).

      6. On the final day, note the number of wells that contain spheroids for each concentration for each population.

        Note: Self-renewing stem cells will maintain the abundance of the stem cell marker, while differentiation will reduce it. Self-renewal in the spheroids can be confirmed by quantifying the abundance of the cells positive for the stem cell marker in the spheroids, and comparing it to that of the original population. This is important to rule out mitochondria driven priming of stem cell differentiation (Chandel et al., 2016).


      Figure 3. Extreme Limiting Dilution Analysis of Four Sorted Populations. A. Example of an advanced spheroid 21 days after seeding acquired using transmitted light on an EVOS fluorescence microscope. Scale bar represents 200 μm. B. Screen capture of the instructions from the ELDA webtool. C. Table showing example data from an ELDA conducted on FACS sorted A2780-CP cells. D. Output of the online ELDA tool plotting input data along with a linear fit used to extrapolate the frequency of sphere-forming cells in each population. E. Table showing estimate and 95% confidence interval of frequency of sphere-forming cells for each population in the A2780-CP cell line. F. Table showing pairwise hypothesis testing of differences in frequencies of sphere-forming cells in each population as output by the online ELDA tool.


    2. Data is analyzed to obtain TIC frequency as follows:

      1. To access the ELDA statistics web tool, open url: http://bioinf.wehi.edu.au/software/elda/.

      2. Enter the population, cells/well, total number of wells, and number of wells containing spheroids into the online ELDA statistics tool (Figures 3B, 3C).

      3. Report the sphere forming capacity and p-value for each population for each day (Figures 3D-3F).

      4. The AldeFluor+ TMRE+ (A+T+) population with maximum frequency of self-renewing and proliferating TICs is deemed as the mitochondria primed TIC population. The A- population has a markedly lower frequency of self-renewing and proliferating TICs, recapitulating the in vivo limiting dilution assay (see Spurlock et al., 2019 for more details).


  5. Example applications of the method to study mitochondrial morphology by confocal microscopy in ovarian TICs:

    Note: We have applied the protocol to characterize specific mitochondrial properties (by confocal microscopy), cell cycle markers (by immunoblotting), and drug sensitivity (by crystal violet staining after drug incubation) within the 4 sorted populations.

    1. Coat 8-well live cell chamber with Geltrex or lineage-appropriate matrix.
    2. Resuspend cell pellets from Step C9 half in normal culture media and half in stem cell media.
    3. Seed cells in live cell chamber to 80% confluence and allow to recover overnight.
    4. Stain cells with MitoTracker Far Red to measure mitochondrial morphology and MitoSox to measure mitochondrial oxidation according to manufacturer’s protocols.
    5. Acquire images according to the MitoGraph section of our protocol (Spurlock et al., 2019 and 2020).
    6. Figures 4A, 4B show the comparisons among the four populations allowed to recover in normal culture media using our metrics for Fission and Fusion-1. Figure 4C shows the relative mitochondrial oxidation of the four populations.


    Figure 4. Application of Method to Study Mitochondrial Morphology of Ovarian TICs . A. Bar graph showing the mean Fission metric of cells from the 4 sorted population. N=18-25. Error Bar shows ± 1SD. P-value is from Student’s t-test. B. Dot plot showing an inverse relationship between Fission and Fusion metrics of each population. C. Bar graph showing the mean MitoSox fluorescence intensity of cells from each population. N = 19-22. Error Bar shows ± 1SD. P-value is from Student’s t-test.

Recipes

  1. TIC media

    Serum-free media base (RPMI or DME/F12)

    1× N1 supplement

    10 μg/ml insulin

    20 ng/ml EGF

    10 ng/ml bFGF

    1% sodium pyruvate

    1% L-glutamine

    1% pen-strep

    Discard unused media after 1 month and make fresh

Acknowledgments

This work was supported by the National Institutes of Health (NIH) [R33ES025662] to B.S. and K.M. This protocol was originally reported in “New quantitative approach reveals heterogeneity in mitochondrial structure-function relations in tumor-initiating cells” published in 2019 in the Journal of Cell Science (Spurlock et al., 2019).

Competing interests

The authors have no competing interests.

References

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简介

[摘要]已经发现各种干细胞都依赖于线粒体的能量学。线粒体在调节正常干细胞和干细胞样肿瘤起始细胞(TICs)自我更新中的作用日益受到人们的重视。我们提出,TIC种群具有由线粒体“引发”自我更新的亚细胞群。使用卵巢癌模型,我们开发了一种协议,可以使用荧光辅助细胞分选(FACS)识别和分离这些“初免”细胞。我们结合活细胞染色剂作为TIC的功能标记物和线粒体跨膜电位,以富集具有更高线粒体电位的TIC(在体外形成)球状体比线粒体电位较低的其他TIC高10倍。该协议可以直接使用或修改以用于各种小区类型。因此,预期该方案对于干细胞群体中线粒体和能量异质性的基本理解是不可侵犯的,并且在再生医学和癌症生物学的转化研究中也可能被证明是有价值的。


背景技术肿瘤中的细胞异质性对癌症治疗提出了严峻的挑战(Magee等,2012)。当某些肿瘤通过自身更新和分化维持肿瘤时,其与肿瘤抗性的肿瘤起始细胞(也称为癌症干细胞)分层地排列在细胞层次的底部(Magee et al。,2012)。肿瘤起始细胞(TICs)的可塑性使其难以鉴定,分离和研究。TICs的能量学的异质性和可塑性才刚刚开始被人们所认识,并且还远远不清楚(Martinez-Outschoorn等人,2017)(Intlekofer和Finley,2019)。此外,TIC的高能状态似乎是肿瘤特异性的,某些TIC依赖于线粒体的高能,并且可以被线粒体抑制剂所消除(Pasto等人,2014; Sancho等人,2015; Viale等人,2015; Martinez -Outschoorn et al。,2017; Spurlock et al。,2019)。

线粒体能量是通过多种生物能参数的复杂生化调节来维持的,其中包括跨线粒体内膜(Δ )维持的线粒体跨膜电位,该电位被消耗以产生ATP,热量或活性氧。期望更高的线粒体跨膜电位将维持更高的ATP合成,具体取决于各种其他参数,例如底物利用率等。以前,已经发现基于线粒体跨膜电位分离的干细胞在干细胞特性方面有所不同(Schieke等,2008; Sukumar等,2016)。最近,我们开发了一种流式细胞协议同时排序细胞Δ  ,使用的红色荧光染料电位TMRE,和功能性TIC标志ALDE的活性海德脱氢酶(ALDH),采用ALDEFLUOR试剂(图1-2) (斯珀洛克等,2019)。使用这种流式细胞仪分选策略,我们能够确定人卵巢TICs的亚群,其自我更新和增殖能力高10倍,这是通过体外有限稀释法测定的。因此,我们的协议可以解决干细胞群体中的线粒体异质性。

随着我们对干细胞能量学的了解增加,线粒体能量学在维持正常干细胞和TICs中的作用得到了更多的赞赏(Viale等人,2015; Chandel等人,2016; Intlekofer和Finley,2019)。成体干细胞很多被大量曲iescent并且被认为是高度糖酵解和释放从静止涉及仍然存在不良understo代谢/高能偏移OD (Margineantu等人,2002; Ito和苏达,2014; Folmes和泰尔齐奇,2016) 。我们提出,具有其他特别的线粒体特性的线粒体跨膜电位特别高的卵巢TICs亚群应为更大的自我更新和增殖能力做好准备(Spurlock等人,2019)。在造血谱系中也观察到了类似的基于线粒体的引发,并且与从静止退出并进入细胞周期进行自我更新和分化有关(Liang et al。,2020)。我们的协议具体隔离这些线粒体引发干细胞(mpSCs)由TIC标志物(ALDH活性)与Δ组合 。Aldh活性是多种肿瘤类型(包括卵巢癌,乳腺癌,宫颈癌,肺癌,肝癌,前列腺癌,骨髓癌,皮肤癌,结肠癌和胰腺癌)的TIC标记物(Marcato等,2011; Toledo-Guzmán等,2019) ;Vassalli,2019年)。我们在人类卵巢癌细胞系中开发了该协议;但是,我们还在源自小鼠异种移植模型以及患者腹水的人卵巢癌细胞中进行了这些检测。在分选的细胞可以研究干细胞或相关方法的其他属性。我们已经研究了使用我们的新的分析排序后细胞线粒体结构和功能特性丝裂因为2 (斯普尔洛克等人,2019) (在图示例URE 3)。该协议是用于从细胞系以及原代细胞群中分离和研究正常和赘生干细胞的简单且有价值的工具。因此,该方案将允许对干细胞群体内的线粒体和能量异质性有基本的了解,并且在再生医学和癌症生物学的转化研究中也可能被证明是有价值的。

关键字:肿瘤启动细胞, 线粒体能量, 干细胞启动, 自我更新, 荧光激活细胞分选术

材料和[R eagents
1.5 ml微量离心管(Eppendorf ,目录号:022363204)
15 ml离心管(Corning,目录号:430790)
5 ml流式细胞仪试管(Falcon,目录号:352003)
96孔超低附着板(Corning,目录号:3474)
8孔Lab-Tek II室盖玻片(Nunc,目录号:155409)
所用细胞的适当细胞培养材料和试剂
DPBS(Sigma,目录号:59331C-1000ML)
胰蛋白酶0.05%(GE Healthcare Life Sciences ,目录号:SH30236.02或类似产品)
AldeFluor套件(StemCell Technologies,目录号:01700)
对于其中ALDH活性不标记干性谱系,我们的协议可以通过包括谱系特异性培养条件和活细胞标记物进行修饰为使用任何谱系(CD133等。),确保最小的或用荧光TMRE没有重叠。


四甲基罗丹明,乙酯,高氯酸盐(TMRE)(Invitrogen,目录号:T669)
所用的A2780卵巢癌细胞系的RPMI培养基(Corning,目录号:15-040-CM)或适当的基础培养基
N1补充剂(Sigma,目录号:N6530-5ML)
胰岛素(Sigma,目录号:10516-5ML)
丙酮酸钠(GE H ealthcare Life Sciences ,目录号:SH30239.01)
L-谷氨酰胺(GE H eththcare Life Sciences ,目录号:SH30034.01)
Pen icillin - S trep tomycin Solution (GE Healthcare Life Sciences,目录号:SV30010)
FGF(Sigma,目录号:F0291-25ug)
EGF(Sigma,目录号:E9644-0.2ug)
TIC媒体(请参阅食谱)


设备


BD FACSAria II或具有类似流式细胞仪的设置和跟踪(CS&T)珠,accudrop校准珠,100 μ米FACS细胞喷嘴和最小的488nm和561 nm激光和用于AldeFlour和26分之57650分之525BP的发射过滤器BP for TMRE
1.5 ml微量离心管和15 ml离心管
带层流的标准生物安全罩
用于细胞培养的标准培养箱保持在37 °C和5%CO 2加湿
具有4 ×和10 ×物镜的光学显微镜
任何共聚焦显微镜555纳米的激光(例如,蔡司LSM700)


软件


BD FACSDiva(版本8.0 .1)(具有自动补偿计算功能)或类似产品,用于流式细胞仪分选和分析


程序


参见图1。


注意:鉴于该方案适用于活细胞,请仔细考虑每个程序所花费的时间,以最大程度地提高程序的严谨性和可重复性。请勿在任何步骤中暂停协议,并执行有效的计划以最大程度地减少每个过程所花费的时间。我们手中每个程序所需的时间如下:


FACS的样品制备:2小时
FACS分离出四个种群:每个样品管2 h。
球状体形成和ELDA:此处使用的细胞谱系为15 d。该时间可能随细胞谱系而变化。






图1 。工作流程的图形表示。通过将细胞置于单细胞悬液中并按照所述方法染色来制备对照管和样品管。使用控制管在分选机上设置参数,然后对四个种群进行分选。mpSCs丰富了双阳性人群。


荧光激活细胞分选(FACS)的样品制备
染色准备:
根据制造商指南重新配制,分装和存放AldeFluor试剂盒和TMRE的试剂。检查制造商协议中AldeFluor试剂的化学性质。
正常培养细胞,直到准备开始实验方案为止。分选前两天,种子细胞在正常生长培养基中的融合程度约为40%,这确保了分选的种群将具有足够的细胞供后续实验使用,而该实验是通过对一千万个细胞进行的模拟实验确定的。
注意:尽管我们开发了针对卵巢癌细胞的方案,但是它可以用于具有适当培养条件和干细胞培养基的任何其他细胞(在后续步骤中)。此外,染色和分选方案也适用于直接从任何组织或肿瘤异种移植物中分离的细胞。可以将分选的细胞直接注射到动物中,以利用体内有限稀释测定法或其他特异性测定法分析干细胞特性。


胰蛋白酶,DPBS,培养基,和ALDEFLUOR缓冲液(从ALDEFLUOR试剂盒),以37等份温暖℃下至少三个H我们之前染色。
用胰蛋白酶消化细胞,然后将其转移到15 ml锥形管中。通过加入胰蛋白酶失活的培养基的等量的细胞悬浮液。彻底移液以确保单细胞悬浮液,以形成均匀的细胞悬浮液。
Ñ OTE:作为写入,该协议是用于细胞系。要修改原代细胞和组织,请使用适当的技术将细胞置于同质的单细胞悬液中,并从步骤A 1e开始。


使用优选方法对来自均质细胞悬液的细胞进行计数(在步骤A1d中)。
首先在室温(RT)下以300 × g浸提细胞悬液10分钟,然后除去上清液并将细胞重悬于3 ml温暖的DPBS中,以洗涤细胞。最后,在室温下以300 × g离心细胞10分钟。
用AldeFluor和TMRE染色:
吸出细胞沉淀物的上清液(在步骤A1f中获得),将细胞重悬在温暖的AldeFluor缓冲液中至2 × 10 6个细胞/ ml的浓度,彻底移液以确保单细胞悬液。
将1.5 ml微量离心管的3个标签标记为“未染色”,“ AldeFluor”和“ TMRE”,以用作补偿控件,以校正AldeFluor试剂和TMRE的发射光谱中的重叠。将第四个1.5 ml微量离心管标记为“ DEAB”,与“ TMRE”管一起,将分别用作AldeFluor和TMRE染色的生物和门控对照。最后,将一根15 ml的锥形管标记为“ AldeFluor + TMRE”,以进行实验样品的分类。
将步骤A 2a中的细胞悬液中的1 × 10 6个细胞(0.5 ml)分别分配到未染色的AldeFluor,TMRE和DEAB管中。将其余的细胞转移到AldeFluor + TMRE管中。
按照制造商的协议用AldeFlour试剂对细胞进行染色:
甲DD 5μ每2升试剂× 10 6在ALDEFLUOR,DEAB和ALDEFLUOR + TMRE管细胞。
抑制ALDH活性在使用5的DEAB管μ每1L × 10 6细胞。
将所有五个试管在37 °C和5%CO 2下孵育1 h(或根据制造商的协议进行优化)。轻拍试管,每15分钟重悬细胞一次。
在室温下以300 × g将所有5个试管中的细胞离心10分钟。
虽然前面的步骤是持续的,准备TMRE在温暖ALDEFLUOR缓冲25nM的溶液。
通过抽吸上清液并将25nM TMRE溶液重悬至2 × 10 6个细胞/ ml,用TMRE对TMRE和AldeFluor + TMRE管中的细胞染色。
在温暖的AldeFluor缓冲液中将未染色,AldeFluor和DEAB管中的细胞重悬至2 × 10 6细胞/ ml。
将试管在37 °C和5%CO 2下孵育15分钟。
在室温下以300 × g离心所有五个试管10分钟。
按照步骤A 2b标记五个5 ml流式细胞仪试管。
通过吸出上清液并将适当重染的细胞转移到等效标记的流式细胞仪管中,并将细胞重悬于AldeFluor缓冲液中,使其浓度为5 × 10 6细胞/ ml。确保没有气泡引入管中。
该TMRE染色方案可有效染色任何给定细胞中的能量活跃的线粒体。但是,应通过将染色的细胞添加到装有活细胞的盖玻片中,使其静置5分钟,并在共聚焦显微镜下观察它们,来证实线粒体的TMRE染色。(图2A)。使用MitoTracker Green染色可能是必要的,以确保染色为线粒体。(Mitra和Lippincott-Schwartz,2010)中描述的协议。如果TMRE未整合到线粒体中,则应通过在给定的细胞密度下改变第一TMRE浓度和孵育时间来重新优化TMRE染色。


用于分选的流式细胞仪数据采集(使用BD FacsAria II优化)
注意:以下预防措施是必要的:


数据采集和分类应在将样品转移至5 ml流式细胞仪的2小时或更短时间内完成。较长的时间可能会导致信号丢失。对于大量的细胞,种子多个板,使得所述第一组单元的可以在两个小时内排序我们的。处理第一组排序时的下一组,依此类推。
应通过运行流式细胞仪设置和跟踪(CS&T)珠子来确保最佳的分选器性能。滴落延迟应使用Accudrop校准珠计算。


可以使用诸如BD FACSDiva(版本8.0.1)之类的软件或分拣器上的类似软件执行数据采集。
附上100 μ米喷嘴在20psi压力进行排序。通过分类,样品管和收集管的温度均保持在4 °C 。S选择以下参数:前向散射(线性标度的FSC)和侧向散射(线性标度的SSC),AldeFluor(Ex:488 nm,Em滤光片:525/50 BP;对数标度)和TMRE(Ex:561)的通道nm,Em滤镜:576/26 BP,对数刻度)。
使用补偿控件调整参数,如下所示:


使用未染色的样品进行以下调整:
调整FSC和SSC的PMT电压,以显示和区分目标目标种群与细胞碎片。
使用参数FSC Area(FSC-A)和SSC Area(SSC-A)(图2B)选出细胞碎片。
栅出使用FSC-高度(FSC-H),FSC-A,SSC宽度(SSC-W)和FSC-高度(SSC-H)在两个随后的栅极(图2C,细胞双联体2 d )。
调整未染色样品的PMT电压,以使TMRE(图2E,左)和AldeFluor(图2E,右)的自发荧光信号峰值低于对数刻度的第二个十年。
使用TMRE染色的样品,调整用于TMRE检测的PMT电压,以确保信号在动态范围内(图2F)。
使用AldeFluor染色的样品,调整用于AldeFluor检测的PMT电压,以确保信号在动态范围内(图2G)。
电压最终确定后,记录来自Unstained,AldeFluor和TMRE管的20,000个事件的数据,并使用自动补偿计算功能来计算荧光信号的补偿并应用于实验。
注意:在建立了分类的可重复性之后,可能不必为每种分类都进行补偿和生物控制。但是,重新引入可控制染色是否已被修改,或者是否处理具有较大固有变化的样本(例如患者样本)。如果要处理患者样品,则必须采取额外的步骤来选通死细胞。






图2.富集和减少线粒体引发的TICs的流式细胞仪策略。一。用TMRE染色的iPSC的显微照片,作为将染料正确地掺入线粒体的例子(左)和坏(右)。比例尺代表5 µm。乙。等高线图显示分别使用FSC-A和SSC-A对上皮性卵巢癌细胞系A2780-CP进行全细胞门控。C.等高线图显示了使用FSC-H和FSC-A对A2780-CP进行的单个细胞的首次选通。D.等高线图显示了使用A2780-CP的SSC-H和SSC-W对单个细胞进行的第二次浇口。E.直方图显示使用TMRE参数(左)和AldeFluor参数(右)在荧光强度的第二个几十年内设置的A2780-CP细胞自动荧光。F.等高线图,显示了仪器动态范围内A2780-CP细胞的TMRE染色。G.等高线图显示了在仪器动态范围内A2780-CP细胞的AldeFluor染色。H.等高线图显示了使用DEAB抑制Aldh活性后的AldeFluor染色。该图标记了AldeFluor的边界–细胞。一,等高线图呈现出四个群体A2780-CP的最终排序门:ALDEFLUOR + TMRE + ,ALDEFLUOR + TMRE - ,ALDEFLUOR - TMRE +,ALDEFLUOR - TMRE - 。


FACS可以分离出四个种群:AldeFluor + TMRE + ,AldeFluor + TMRE – ,AldeFluor – TMRE +,AldeFluor – TMRE –
在执行步骤1C之后,使用生物/门控控件如下设置分类门:
记录20条,用DEAB管和TMRE管000事件。
对于ALDEFLUOR组阈值+ (图URE 2H,红色线)和TMRE + (图2F,红线)。
记录20 ,使用ALDEFLUOR + TMRE 000事件染色的样品。
每个四分位数内周围绘制不同的种群分配门如图URE 1H(黑色矩形)用于排序的以下细胞群体:ALDEFLUOR + TMRE + ,ALDEFLUOR + TMRE - ,ALDEFLUOR - TMRE +,ALDEFLUOR - TMRE - 。
注意:清除碎片足以排除我们细胞系中的死细胞,获得单个细胞悬液后的存活率通常超过90%。其他谱系的新鲜组织和细胞可能需要生存力染色以排除死细胞。污渍必须与TMRE荧光兼容(例如,通过DAPI排除门控活细胞)。


设置流式细胞仪,以记录门控预分类样品中的100,000个事件。使用BD FACSDiva(版本8.0.1)软件确定每个群体中细胞的百分比。AldeFluor + TMRE +群体富含线粒体引发的TIC。
对于要分类的人群,将0.5 ml无菌培养基或适当的干细胞培养基添加到四个5 ml流式细胞仪收集管中,并在试管上贴上“ AldeFluor + TMRE + ”,“ AldeFluor + TMRE – ”,“ AldeFluor – TMRE + ”和“ AldeFluor – TMRE – ”。将收集管放置在FACSAria中对应于在管上标记的种群的分类门(图2I)的位置。
注意小号:


TIC培养基或干细胞培养基的组成在谱系之间可能有所不同。因此,使用适当的干细胞培养基进行球体测定。
如果FACS仪器的位置与样品制备的位置不同,请在无菌容器中将转移细胞,收集管,额外的流式细胞仪以及AldeFluor Buffer和细胞培养基的等分试样置于RT无菌容器中,避免暴露于光下。
在此处提供的示例细胞中,TMRE荧光的分布是双峰的,易于区分阳性和阴性群体。如果分布不是双峰分布,则负数总体应为正态分布的最低10%,正数则应为正态分布的最高10%。
使用100 μ在20psi压力米喷嘴进行排序。通过分类,样品管和收集管的温度均保持在4 °C 。保持流速为每秒9,000到10,000个事件的阈值速率。在加载到样品端口之前,以中等速度涡旋样品。在整个分类过程中,保持样品搅拌速度为200 rpm。
允许分类继续进行,直到达到每个群体的目标细胞数为止,并根据需要更换收集管。
将分选的细胞保持在4 °C,直到随后的实验需要为止。
在接种或收获分类细胞之前,将细胞转移至标记的15 ml锥形管中,并在4 °C下以300 × g离心10分钟。
吸出上清液并将细胞重悬于DPBS或所需的细胞培养基中。在4 °C下以300 × g离心10分钟,然后吸出上清液。
视情况将这些细胞沉淀或细胞悬液用于进一步的实验。


极端极限稀释分析(ELDA)(Hu和Smyth,2009),用于从分类种群形成的球体中定量TIC频率。
注意:尽管这可以是可选的,但我们建议在任何新的谱系或细胞类型中首次使用分选方案时进行此测定。这是因为按此测定法量化的,旨在最大程度自我更新和增殖的分选种群可能与此处所示的卵巢癌系统相似,也可能不相似。也可以使用异种移植物进行体内有限稀释测定法来代替该体外测定法。


椭球的形成如下:
根据分选的细胞数,将每个种群重悬于干细胞培养基中,使其浓度为10 4个细胞/ ml(最终体积至少为3 ml)。
连续稀释上述悬浮液的一部分,以10 3 ,10 2和10个细胞/ ml。
注:10倍的每个稀释物可以通过添加300来实现μ每个连续细胞悬液l至2.7 ml的干细胞培养基在15ml锥形管中。在洗涤步骤后,我们总是使用连续稀释接种细胞,该稀释基于通过台盼蓝排除法对活细胞计数。但是,也可以通过将所需数量的细胞直接分选到96孔超低附着板的每个孔中来完成接种。


分发至少24个孔中的每个细胞悬浮液在一个96孔超低附着板(100 μ升/孔)。每个细胞浓度使用的总孔数越多,测定的统计能力越高。将板放在细胞培养箱中。在图3的示例实验中,我们使用了4个板,每个种群一个。在每个平板中,我们播种24孔,每个细胞1孔; 24孔,每个细胞10孔; 24孔,每个细胞100孔; 24孔,每个细胞10 3个细胞。
每隔第二天,使用光学显微镜的10倍物镜检查每个孔是否形成具有紧密边缘的致密球体(图3A)。
每隔第二天加标干细胞培养基体积的10%的第二次加标细胞,直到在阳性对照组中检测到明显的带有紧边缘的肿瘤球(图3A);在示例中,提供的Aldh +(A +)组为阳性对照,而Aldh-(A-)组为阴性对照(图3)。
在最后一天,请注意每个人口每个浓度的包含球状体的孔的数量。
注意:自我更新的干细胞将保持干细胞标志物的丰度,而分化则会减少它。可以通过对球体中干细胞标记阳性的细胞丰度进行定量并将其与原始种群进行比较,来确认球体的自我更新。这对于排除线粒体驱动的干细胞分化引发很重要(Chandel et al。,2016)。






图3.四个分类种群的极限稀释度分析。A.在EVOS荧光显微镜上使用透射光获得种子后21天的高级球状体的实例。比例尺条为200 μ米。B. ELDA网络工具中的指令的屏幕截图。C.表显示了来自ELDA的示例数据,该数据是在FACS分选的A2780-CP电池上进行的。D.在线ELDA工具的输出,它绘制输入数据以及用于推断每个群体中形成球体的细胞的频率的线性拟合。E.表显示了对于A2780-CP细胞系中每个群体的球形成细胞的频率的估计和频率的95%置信区间。F.该表显示了在线ELDA工具输出的每个群体中形成球形细胞的频率差异的成对假设检验。


对数据进行分析以获得TIC频率,如下所示:
要访问ELDA统计Web工具,请打开URL:http : //bioinf.wehi.edu.au/software/elda/。
输入的人口,个细胞/孔,总的孔的数目,以及包含球状体进入在线ELDA统计工具井的数量(图小号3B,3 C)。
报告每天每个人口的球形成能力和p值(图s 3D- 3 F)。
具有自我更新和增殖的TIC频率最大的AldeFluor + TMRE + (A + T + )种群被认为是线粒体引发的TIC种群。A族群体具有自我更新和增殖的TIC的频率显着降低,概括了体内有限稀释试验(更多细节参见Spurlock等人,2019)。 


通过共聚焦显微镜在卵巢TICs中研究线粒体形态的方法的示例应用:
注意:我们已应用该协议在4个分类的群体中表征特定的线粒体特性(通过共聚焦显微镜),细胞周期标记(通过免疫印迹)和药物敏感性(通过药物孵育后的结晶紫染色)。


在8孔活细胞室中涂上Geltrex或适合谱系的基质。
将步骤C9中的细胞沉淀重悬在正常培养基中,一半在干细胞培养基中。
活细胞室中的种子细胞汇合至80%,并使其恢复过夜。
根据制造商的规程,使用MitoTracker远红染色细胞以测量线粒体形态,并使用MitoSox染色以测量线粒体氧化。
根据我们的协议的MitoGraph部分获取图像(Spurlock等人,2019和2020)。
图小号4A,4 B示出允许USI在正常培养基中回收,种群之间的比较纳克我们的裂变和聚变-1指标。图4C显示了这四个群体的相对线粒体氧化。






图4.该方法在研究卵巢TICs的线粒体形态中的应用。A.条形图显示了来自4个排序群体的细胞的平均裂变度量。N = 18-25。错误栏显示± 1SD。P -值是小号tudent的牛逼-测试。B.点图显示了每个人群的裂变和融合指标之间的反比关系。C.条形图显示了来自每个群体的细胞的平均MitoSox荧光强度。N = 19-22。误差棒显示为± 1SD 。P -值是小号tudent的牛逼-测试。


菜谱


TIC媒体
无血清培养基(RPMI或DME / F12)


1 × N1补品


10 μ微克/毫升胰岛素


20 ng / ml EGF


10 ng / ml bFGF


1%丙酮酸钠


1%L-谷氨酰胺


1%链球菌


1个月后丢弃未使用的介质并重新制作


致谢


这项工作得到了BS和KM的国立卫生研究院(NIH)[R33ES025662]的支持。该协议最初发表在“新的定量方法揭示了肿瘤起始细胞中线粒体结构-功能关系的异质性”中,该研究于2019年在《华尔街日报》上发表细胞科学》(Spurlock等人,2019)。


利益争夺


作者没有竞争利益。


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引用:Spurlock, B., Hanumanthu, V. S. and mitra, K. (2021). Strategy of Isolating ‘Primed’ Tumor Initiating Cells Based on Mitochondrial Transmembrane Potential. Bio-protocol 11(5): e3945. DOI: 10.21769/BioProtoc.3945.
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