High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS

Materials and Reagents

1. Falcon 25 cm² rectangular canted neck cell culture flask with plug seal screw cap (Corning, catalog number: 353082)

2. Nunc EasYDish dishes 100 mm (Thermo Fisher Scientific, catalog number: 150466)

3. Primaria 25 cm² rectangular canted neck cell culture flask with vented cap (Corning, catalog number: 353808)

4. Safe lock tubes 2.0 mL (Eppendorf, catalog number: 3-7353-04)

5. Tube, 15 mL, PP, 17/120 mm, conical bottom, cellstar, blue screw cap, natural, graduated, writing area, sterile, 5 psc./bag, triple packed (Greiner Bio-One, catalog number: 188271)

6. Pipette, 10 mL, graduated 1/10 mL, sterile, paper-plastic packaging, single packed (Greiner Bio-One, catalog number: 607180)

7. Pipette, 25 mL, graduated 2/10 mL, sterile, paper-plastic packaging, single packed (Greiner Bio-One, catalog number: 760180)

8. 10 μL, long filter tip, graduated, system rack (PP), sterilized (WATSON, catalog number: 1252P-207CS)

9. 20 μL, hyper filter tip, refill plate, sterilized (WATSON, catalog number: 127-20S)

10. 200 μL, hyper filter tip, refill plate, sterilized (WATSON, catalog number: 0127-20S)

11. 1,000 μL, long filter tip, graduated, refill plate, sterilized (WATSON, catalog number: 126-1000S)

12. Falcon 5 mL round bottom polystyrene test tube, with snap cap, sterile (Corning, catalog number: 352058)

13. Falcon 5 mL round bottom polystyrene test tube, with cell strainer snap cap (Corning, catalog number: 352235)

14. Stericup quick release-GP sterile vacuum filtration system (Millipore, catalog number: S2GPU01RE)

15. A375 [American Type Culture Collection (ATCC), catalog number: CRL-1619]

16. C2C12 (ATCC, catalog number: CRL-1772)

17. H1944 (ATCC, catalog number: CRL-5907)

18. H2009 (ATCC, catalog number: CRL-5911)

19. H2122 (ATCC, catalog number: CRL-5985)

20. H441 (ATCC, catalog number: HTB-174)

21. HEK293 (ATCC, catalog number: CRL-1573)

22. HEK293T (ATCC, catalog number: CRL-3216)

23. HeLa S3 (RIKEN BioResource Research Center, RCB1525)

24. Alexa-Fluor 647 Anti-Tom20 antibody (Abcam, catalog number: ab209606, stored at 4°C)

25. Anisomycin (from Streptomyces griseolus) (Chem-Impex International, catalog number: 00466, stored at -20°C)

26. 10× Click-iT cell reaction buffer (Thermo Fisher Scientific, catalog number: C10269, stored at 4°C)

27. Click-iT reaction buffer additive (Thermo Fisher Scientific, catalog number: C10269, stored at -20°C)

28. Copper (II) sulfate (CuSO₄) (Thermo Fisher Scientific, catalog number: C10269, stored at 4°C)

29. Cy3-azide (Jena Bioscience, catalog number: CLK-046, stored at -20°C)

30. Digitonin (Nacalai Tesque, catalog number: 19390-91, stored at room temperature)

31. DMEM, high glucose, GlutaMAX supplement (Thermo Fisher Scientific, catalog number: 10566-016, stored at 4°C)

32. DMEM, high glucose, no glutamine, no methionine, no cystine (Thermo Fisher Scientific, catalog number: 21013-024, stored at 4°C)

33. Dimethyl sulfoxide (DMSO), nuclease and protease tested (Nacalai Tesque, catalog number: 09659-14, stored at room temperature)

34. L-homopropargylglycine (HPG) (Jena Bioscience, catalog number: CLK-1067, stored at -20°C)

35. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524, stored at -20°C)

36. 1 mol/L HEPES-KOH buffer solution (pH 7.5) (Nacalai Tesque, catalog number: 15639-84, stored at room temperature)

37. Intercept (TBS) blocking buffer (LI-COR, catalog number: 927-60001, stored at 4°C)

38. 200 mmol/L L-alanyl-L-glutamine solution (100×) (Nacalai Tesque, catalog number: 04260-64, stored at 4°C)

39. L-cystine dihydrochloride, animal-free (Nacalai Tesque, catalog number: 13003-12, stored at room temperature)

40. 1 mol/L magnesium chloride solution (MgCl₂), sterile-filtered (Nacalai Tesque, catalog number: 20942-34, stored at room temperature)

41. 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque, catalog number: 09154-85, stored at 4°C)

42. 5 mol/L sodium chloride solution (NaCl) (Nacalai Tesque, catalog number: 06900-14, stored at room temperature)

43. Sucrose, ultra-pure (Wako, catalog number: 198-13525, stored at room temperature)

44. Triton X-100 (Nacalai Tesque, catalog number: 12967-32, stored at room temperature)

45. Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, catalog number: 25300-054, stored at 4°C)

46. UltraPure DNase/RNase-Free distilled water (Thermo Fisher Scientific, catalog number: 10977-015, stored at room temperature)

47. Bovine serum albumin (Sigma-Aldrich, catalog number: A4919)

48. Met-free DMEM (500 mL) (see Recipes)

49. 100 mg/mL anisomycin (250 µL) (see Recipes)

50. 100 mM HPG (6.11 mL) (see Recipes)

51. Met-free DMEM with anisomycin and HPG (10 mL) (see Recipes)

52. Met-free DMEM with anisomycin (10 mL) (see Recipes)

53. 0.5% digitonin (10 mL) (see Recipes)

54. Mitochondria-protective buffer (5 mL) (see Recipes)

55. Mitochondria-protective buffer with 0.0005% digitonin (5 mL) (see Recipes)

56. 10% Triton X-100 (40 mL) (see Recipes)

57. PBS with 0.1% Triton X-100 (0.5 mL) (see Recipes)

58. 5 mM Cy3-azide (370 µL) (see Recipes)

59. 50 µM Cy3-azide (100 µL) (see Recipes)

60. Click-reaction mixture (1 mL) (see Recipes)

61. Intercept (TBS) blocking buffer with Alexa-Fluor 647 Anti-Tom20 antibody (101 µL) (see Recipes)

62. FACS buffer (see Recipes)

Equipment

1. High-speed micro centrifuge (himac, model: CF16RN)

2. High-speed refrigerated micro centrifuge (TOMY, model: MDX-310)

3. Swing rotor (himac, model: T4SS31)

4. Swing rack (TOMY, model: SAR015-24)

5. CO₂ incubator (PHCbi, model: MAC-170AIC)

6. Pipet-Aid XP2 (Drummond Scientific Company, catalog number: 4-040-501)

7. Pipetman P-10 (Gilson, catalog number: F144802)

8. Pipetman P-20 (Gilson, catalog number: F123600)

9. Pipetman P-200 (Gilson, catalog number: F123601)

10. Pipetman P-1000 (Gilson, catalog number: F123602)

11. BD FACSMelody [BD, 3-laser (488, 640, and 561 nm), 8-color (2-2-4) configuration)]

12. CellDrop BF (DeNovix, CellDrop BF-UNLTD)

Software

1. BD FACSChorus (BD FACSMelody operating software)

2. FlowJo (BD, v10.8.1)

3. R version 4.1.1 (R Development Core Team, https://www.r-project.org )

Procedure

Note: When the cells are centrifuged, use swing rotors.

1. Cell culture

   1. Culture cells to 70%–80% confluency with 5 mL of medium in a humidified incubator with 5% CO₂ at 37°C. The media and flask used for each cell line in our experiments are described below.

      Medium and flasks:

      
      

      Cell type	Media	Flask
      C2C12	DMEM, high glucose, GlutaMAX supplement with 10% FBS	Falcon T25 flask
      HeLa S3	Falcon T25 flask
      HEK293	Falcon T25 flask
      HEK293T	Falcon T25 flask
      A375	Falcon T25 flask
      H2009	Falcon T25 flask
      H2122	RPMI 1640 medium with 10% FBS	Primaria T25 flask
      H1944	Falcon T25 flask
      H441	Falcon T25 flask

   
   

   2. Aspirate the medium from the flask and add 3 mL of PBS slowly.

   3. Aspirate PBS from the flask and add 1 mL of trypsin-EDTA.

   4. Incubate the cells in a humidified incubator with 5% CO₂ for 5 min at 37°C.

   5. Add 5 mL of the medium described in the table above and detach cells from the flask by pipetting.

   6. Transfer the cell suspension to a 15 mL tube.

   7. Centrifuge the cells at 800 × g for 3 min at 25°C.

   8. Aspirate the supernatant and resuspend the cell pellet with 5 mL of the medium described in the table above.

   9. Count the cell number by CellDrop.

      Note: The cell suspension should be at 0.5 × 10⁶ –2 × 10⁶ cells/mL.

   10. Dilute the cell suspension to 2 × 10⁵ cells/mL with the medium described in the table above.

   11. Seed 10 mL of cell suspension (2 × 10⁵ cells/mL) in a 10 cm dish.

       Note: The following cultures should be prepared: one culture for the tested sample and another for the negative control for FACS analysis.

   12. Incubate the cells in a humidified incubator with 5% CO₂ overnight at 37°C.




2. HPG labeling

   13. For the sample, aspirate the medium from one of the 10 cm dishes and add 10 mL of Met-free DMEM (see Recipes).

   14. Aspirate the medium and add 10 mL of Met-free DMEM with anisomycin and HPG (see Recipes) to the 10 cm dish. For the negative control, use DMEM with anisomycin (see Recipes).

       Note: Anisomycin is used to shut off cytosolic protein synthesis.

   15. Incubate the 10 cm dish in a humidified incubator with 5% CO₂ for 3 h at 37°C.




3. Fixation and permeabilization

   16. Aspirate the medium from the 10 cm dish and add 10 mL of PBS.

   17. Aspirate the PBS and add 1.5 mL of trypsin-EDTA.

   18. Incubate the cells in a humidified incubator with 5% CO₂ for 5 min at 37°C.

   19. Transfer the cell suspension to a 2 mL tube.

   20. Centrifuge at 300 × g for 3 min at 25°C.

   21. Aspirate the supernatant and resuspend the cell pellet with 0.5 mL of cold PBS.

   22. Centrifuge the cells at 300 × g for 3 min at 25°C.

   23. Aspirate the supernatant and resuspend the cell pellet with 0.5 mL of mitochondria-protective buffer with 0.0005% digitonin (see Recipes) at room temperature.

   24. Incubate the cells for 5 min at 25°C.

   25. Centrifuge the cells at 300 × g for 3 min at 25°C.

   26. Discard the supernatant with a pipette and resuspend the cell pellet with 0.5 mL of ice-cold 4% paraformaldehyde phosphate buffer solution on ice.

   27. Incubate the cells for 15 min on ice.

   28. Centrifuge the cells at 300 × g for 3 min at 25°C.

   29. Discard the supernatant with a pipette and resuspend the cell pellet with 0.5 mL of PBS with 0.1% Triton X-100 (see Recipes) at room temperature.

   30. Incubate the cells for 5 min at 25°C.

   31. Centrifuge the cells at 1,000 × g for 3 min at 25°C.

   32. Discard the supernatant and proceed to the “Click reaction” immediately.




4. Click reaction

   33. Resuspend the cell pellet with 250 µL of click-reaction mixture (see Recipes) at room temperature.

   34. Incubate the cells for 30 min at 25°C.

   35. Centrifuge at 1,000 × g for 3 min at 25°C.

   36. Discard the supernatant with a pipette and proceed to the “Tom20 immunostaining” immediately.




5. Tom20 immunostaining

   37. Resuspend the cell pellet with 0.5 mL of intercept (TBS) blocking buffer at room temperature.

   38. Centrifuge at 1,000 × g for 3 min at 25°C.

   39. Discard the supernatant with a pipette and resuspend the cell pellet with 100 µL of ice-cold intercept (TBS) blocking buffer with Alexa-Fluor 647 Anti-Tom20 antibody (see Recipes) on ice.

       Note: For negative control of Tom20 immunostaining, use ice-cold intercept (TBS) blocking buffer (omitting Alexa-Fluor 647 Anti-Tom20 antibody).

   40. Incubate the cells for 1 h on ice.

   41. Centrifuge at 1,000 × g for 3 min at 2°C.

   42. Discard the supernatant with a pipette and resuspend the cell pellet in 0.5 mL of cold PBS.

   43. Repeat steps E41–42 twice (for a total of three washes).

   44. Resuspend the cell pellet in 0.5 mL of cold FACS buffer (see Recipes).

   45. Proceed to “FACS preparation and analysis” immediately.




6. FACS preparation and analysis

   46. Filter the cell suspension with a cell strainer and collect the cells in a 5 mL round-bottom polystyrene test tube.

   47. Transfer the cells that passed through the filter to a new 5 mL round-bottom polystyrene test tube and preserve in ice until measurement.

   48. Place the 5 mL round-bottom polystyrene test tube containing the negative control (neither HPG-Cy3 nor Tom20-AF647 labeling) on the BD FACSMelody and start the flow.

   49. Set appropriate photomultiplier tube (PMT) voltages for forward scatters (FSCs) and side scatters (SSCs) with BD FACSChorus so that the signal lies within the detectable range. Then, gate a cell population as “Gate: Cells” in BD FACSChorus.

   50. To discriminate doublets, set “Gate: Singlet” according to the values of FSC-A/FSC-H and FSC-H/FSC-W in FlowJo.

   51. Set the PMT voltages for HPG-Cy3 labeling [561 nm laser, PE filter set (582/15 filter and 582 LP mirror); alternatively, 488 nm laser, PE filter set (586/42 filter and 560 LP)] and Tom20-AF647 labeling [640 nm laser, APC filter set (660/10 filter and 660/10 mirror)] in BD FACSChorus so that the signal is shown in approximately 100.

   52. Replace the 5 mL round-bottom polystyrene test tube containing the sample with the one containing the negative control in BD FACSMelody and start the flow.

   53. Record the data up to 10,000 events in “Gate: Singlet” and then export all the data at least including FSC-A, SSC-A, PE-A, and APC-A as an FCS file.

Data analysis

1. Load the FCS file in FlowJo.

2. Readjust the gating to discriminate the dead cells, doublet, and triplet as a new population: “New singlet population.”

   Export “New singlet population” as a csv file containing APC-A (Tom20-AF647 signal) and PE-A (HPG-Cy3 signal).

   Note: The representative data shown inFigure 1are provided in representative csv files (“HEK293_WithoutHPG,” “HEK293_WithHPG.csv,” “H441#2.csv,” and “H2122#2.csv”) in the Supplemental material .

3. Load the csv file to R and run “Mito-FUNCAT_FACS_script_Fig1AB.R” or “Mito-FUNCAT_FACS_script_Fig1CD.R” (found in Supplemental items) to conduct the following steps.

4. Extract 10,000 rows from the top of the csv file.

5. Remove rows containing fluorescence intensity less than 0.

6. Normalize the HPG-Cy3 value by the Tom20-AF647 value for each row.

7. Visualize the distribution of AF647-normalized Cy3 values in the density plot (seeFigure 1for representative data).

   Note: To determine statistical significance between samples, we typically used the MannWhitney U test.

Figure 1. Distribution of Tom20-AF647 (mitochondrial mass)–normalized HPG-Cy3 (mitochondrial translation) signals. (A–D) The Cy3-conjugated nascent peptide in mitochondria was normalized to AF647-labeled Tom20 abundance for HEK293 (A and B), H411 (C), and H2122 (D). For A, HPG was omitted from the medium as the negative control. The vertical lines represent the median of the distributions. The original data (“HEK293_WithoutHPG,” “HEK293_WithHPG.csv,” “H441#2.csv,” and “H2122#2.csv”) and scripts (“Mito-FUNCAT_FACS_script_Fig1AB.R” and “Mito-FUNCAT_FACS_script_Fig1CD.R”) that were used to generate these graphs are provided in the Supplemental material .

Recipes

1. Met-free DMEM (500 mL)

   Reagent	Final concentration	Amount
   L-cystine dihydrochloride	48 µg/mL	24 mg
   200 mM L-alanyl-L-glutamine solution	4.08 mM	10.2 mL
   FBS	10%	50 mL
   DMEM (4.5 g/L D-glucose, no L-glutamine, no sodium pyruvate, no L-methionine and L-cystine)	n/a	439.8 mL




2. 100 mg/mL anisomycin (250 µL)

   Reagent	Final concentration	Amount
   Anisomycin	100 mg/mL	25 mg
   DMSO	n/a	250 µL




3. 100 mM HPG (6.11 mL)

   Reagent	Final concentration	Amount
   HPG	100 mM	100 mg
   DMSO	n/a	6.11 mL




4. Met-free DMEM with anisomycin and HPG (10 mL)

   Reagent	Final concentration	Amount
   Met-free DMEM	n/a	10 mL
   100 mg/mL anisomycin	100 µg/mL	10 µL
   100 mM HPG	100 µM	10 µL

   Note: Prepare before use.




5. Met-free DMEM with anisomycin (10 mL)

   Reagent	Final concentration	Amount
   Met-free DMEM	n/a	10 mL
   100 mg/mL anisomycin	100 µg/mL	10 µL

   Note: Prepare before use.




6. 0.5% digitonin (10 mL)

   Reagent	Final concentration	Amount
   Digitonin	0.5%	50 mg
   RNase-free water	n/a	10 mL




7. Mitochondria-protective buffer (5 mL)

   Reagent	Final concentration	Amount
   Sucrose	10% (w/v)	0.5 g (corresponds to 370 μL)
   5 M NaCl	10 mM	10 µL
   1 M MgCl2	5 mM	20 µL
   1 M HEPES-KOH pH 7.5	10 mM	50 µL
   RNase-free water	n/a	4.55 mL




8. Mitochondria-protective buffer with 0.0005% digitonin (5 mL)

   Reagent	Final concentration	Amount
   Mitochondria-protective buffer	n/a	5 mL
   0.5% digitonin	0.0005%	5 µL




9. 10% Triton X-100 (40 mL)

   Reagent	Final concentration	Amount
   Triton X-100	10%	4 mL
   RNase-free water	n/a	36 mL




10. PBS with 0.1% Triton X-100 (0.5 mL)

    Reagent	Final concentration	Amount
    PBS	n/a	0.5 mL
    10% Triton X-100	0.1%	5 µL




11. 5 mM Cy3-azide (370 µL)

    Reagent	Final concentration	Amount
    Cy3-azide (MW: 539.35)	5 mM	1 mg
    DMSO	n/a	370 µL




12. 50 µM Cy3-azide (100 µL)

    Reagent	Final concentration	Amount
    5 mM Cy3-azide	50 µM	1 µL
    RNase-free water	n/a	99 µL

    Note: Prepare before use.




13. Click-reaction mixture (1 mL)

    Reagent	Final concentration	Amount
    10× Click-iT cell reaction buffer	1×	100 µL
    100 mM CuSO4	2 mM	20 µL
    Click-iT reaction buffer additive	n/a	100 µL
    50 µM Cy3-azide	1 µM	20 µL
    RNase-free water	n/a	760 µL

    Note: Prepare before use.




14. Intercept (TBS) blocking buffer with Alexa-Fluor 647 Anti-Tom20 antibody (101 µL)

    Reagent	Final concentration	Amount
    Intercept (TBS) blocking buffer	n/a	100 µL
    Alexa-Fluor 647 Anti-Tom20 antibody	n/a	1 µL

    Note: Prepare before use and keep on ice.




15. FACS buffer

    Reagent	Final concentration	Amount
    PBS	n/a	100 mL
    Bovine serum albumin	3%	3 g

    Note: Prepare before use.

Acknowledgments

We are grateful to Yusuke Kimura, Taisei Wakigawa, Yasuhiro Ikegami, Kenji Ohtawa for their constructive advice on the experiments and to all the members of the Iwasaki laboratory for their critical reading of the manuscript. We also thank the Komaba Analysis Core, Institute of Industrial Science, The University of Tokyo, for FACS analysis. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (JP20H05784 to S.I.; JP20H05786 to Y.I.) and the Japan Agency for Medical Research and Development (AMED) (JP21gm1410001 to S.I. and Y.I.). H.S. was a RIKEN Junior Research Associate (JRA).

This protocol was derived from an original paper (Kimura et al., 2022) and the manufacturer's protocol for the Click-it Cell Reaction Buffer Kit .

Competing interests

The authors declare no competing interests.

Ethics

No human or animal subjects were included in this study.

References

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