Optimized Isolation of Lysosome-Related Organelles from Stationary Phase and Iron-Overloaded Chlamydomonas reinhardtii Cells

Materials and reagents

Reagents

1. NH₄Cl (Sinopharm Chemical Reagent, CAS number: 12125-02-9)

2. MgSO₄·7H₂O (Sinopharm Chemical Reagent, CAS number: 10034-99-8)

3. CaCl₂·H₂O (Sinopharm Chemical Reagent, CAS number: 10035-04-8)

4. Tris base (Genview, CAS number: BT350-500G)

5. KH₂PO₄ (Sinopharm Chemical Reagent, CAS number: 7778-77-0)

6. K₂HPO₄ (Sinopharm Chemical Reagent, CAS number: 7758-11-4)

7. Na₂EDTA·2H₂O (Sinopharm Chemical Reagent, CAS number: 6381-92-6)

8. (NH₄)₆Mo₇O₂₄ (Sinopharm Chemical Reagent, CAS number: 12054-85-2)

9. ZnSO₄·7H₂O (Sinopharm Chemical Reagent, CAS number: 7446-20-0)

10. H₃BO₃ (Sinopharm Chemical Reagent, CAS number: 10043-35-3)

11. MnCl₂·4H₂O (Sinopharm Chemical Reagent, CAS number: 13446-34-9)

12. CoCl₂·6H₂O (Sinopharm Chemical Reagent, CAS number: 7791-13-1)

13. CuSO₄·5H₂O (Sinopharm Chemical Reagent, CAS number: 7758-99-8)

14. FeSO₄·7H₂O (Sinopharm Chemical Reagent, CAS number: 7782-63-0)

15. KOH (Sinopharm Chemical Reagent, CAS number: 1310-58-3)

16. Glacial acetic acid (Sinopharm Chemical Reagent, CAS number: 64-19-7)

17. NH₄Cl (MACKLIN, CAS number: 12125-02-9)

18. CaCl₂·2H₂O (MACKLIN, CAS number: 10035-04-8)

19. MgSO₄·7H₂O (MACKLIN, CAS number: 10034-99-8)

20. K₂HPO₄ (MACKLIN, CAS number: 7758-11-4)

21. KH₂PO₄ (MACKLIN, CAS number: 7778-77-0)

22. Acetic acid (MACKLIN, CAS number: 64-19-7)

23. EDTA Na₂·2H₂O (Genview, CAS number: 6381-92-6)

24. (NH₄)₆Mo₇O₂₄·4H₂O (MACKLIN, CAS number: 12027-67-7)

25. ZnSO₄·7H₂O (MACKLIN, CAS number: 7446-20-0)

26. MnCl₂·4H₂O (MACKLIN, CAS number: 13446-34-9)

27. FeCl₃·6H₂O (MACKLIN, CAS number: 10025-77-1)

28. Na₂CO₃ (MACKLIN, CAS number: 497-19-8)

29. CuCl₂·2H₂O (MACKLIN, CAS number: 10125-13-0)

30. HCl (Sinopharm Chemical Reagent, CAS number: 7647-01-0)

31. EGTA (Genview, CAS number: 67-42-5)

32. KCl (Sinopharm Chemical Reagent, CAS number: 7447-40-7)

33. MgCl₂ (Sinopharm Chemical Reagent, CAS number: 7786-30-3)

34. Sucrose (Biofroxx, CAS number: 57-50-1)

35. OptiPrep density gradient medium (Sigma-Aldrich, CAS number: 92339-11-2)

36. DTT (Blotopped, CAS number: 3483-12-3)

37. Protease inhibitor cocktail (Sigma, CAS number: P9599)

1. TAP medium (see Recipes)

2. Revised TAP (see Recipes)

3. IB buffer (see Recipes)

4. OptiPrep gradient (see Recipes)

5. 2 M DTT (see Recipes)

1. Tris-acetate-phosphate (TAP) medium

   Reagent	Final concentration	Quantity or Volume
   TAP salts (100×)	1% (v/v)	10 mL
   Tris base (100×)	1% (v/v)	10 mL
   Phosphate buffer (1,000×)	0.1% (v/v)	1 mL
   Hutner trace elements (1,000×)	0.1% (v/v)	1 mL
   Glacial acetic acid	0.1% (v/v)	1 mL
   Milli-Q Water		Dilute with H2O to a final volume of 1 L
   Total		1 L

   
   

   Stocks for TAP medium:

   1. TAP salts (100×)

      Reagent	Final concentration	Quantity or Volume
      NH4Cl	0.7 M	37.5 g
      MgSO4·7H2O	40 mM	10 g
      CaCl2·H2O	30 mM	5 g
      Milli-Q H2O		Dilute with H2O to a final volume of 1 L
      Total		1 L

   
   

   2. Tris base (100×)

      Reagent	Final concentration	Quantity or Volume
      Tris base	2 M	242 g
      Milli-Q H2O		Dilute with H2O to a final volume of 1 L
      Total		1 L

   
   

   3. Phosphate buffer (1,000×)

      Reagent	Final concentration	Quantity or Volume
      KH2PO4	0.4 M	27 g
      K2HPO4	0.62 M	54 g
      Milli-Q H2O		Dilute with H2O to a final volume of 500 mL
      Total		500 mL

   
   

   4. Hutner trace elements

      Note: Store at 4 °C.

      Reagent	Quantity or Volume
      Na2EDTA·2H2O	55.36 g
      (NH4)6Mo7O24·4H2O	1.108 g
      ZnSO4·7H2O	22 g
      H3BO3	11.4 g
      MnCl2·4H2O	5.06 g
      CoCl2·6H2O	1.61 g
      CuSO4·5H2O	1.57 g
      FeSO4·7H2O	4.99 g
      Milli-Q H2O	Dilute with H2O to a final volume of 1 L
      Total	1 L

      Dissolve 55.36 g Na₂EDTA·2H₂O in 250 mL of H₂O and gradually add KOH with heating to accelerate the dissolution. Dissolve 1.108 g of (NH₄)₆Mo₇O₂₄·4H₂O in 50 mL of H₂O. Dissolve 22 g of ZnSO₄·7H₂O in 100 mL of H₂O. Dissolve 11.4 g of H₃BO₃ in 200 mL of H₂O with heating. Dissolve 5.06 g of MnCl₂·4H₂O in 50 mL of H₂O. Dissolve 1.61 g of CoCl₂·6H₂O in 50 mL of H₂O. Dissolve 1.57 g of CuSO₄·5H₂O in 50 mL of H₂O. Dissolve 4.99 g of FeSO₄·7H₂O in 50 mL of H₂O before mixing to avoid oxidation.

      Once all the solutions mentioned above are prepared, mix them, excluding Na₂EDTA. Boil the mixture, then add the Na₂EDTA solution. The mixture will turn green. Ensure all reagents are completely dissolved, then cool the solution to 70 °C and maintain this temperature. Adjust the pH to 6.7 using KOH (calibrate the pH meter at 70 °C; NaOH is unsuitable for this purpose). After adjusting the pH, dilute the solution to a final volume of 1 L, seal the conical flask with cotton, and let it stand for 1–2 weeks, shaking daily. The solution will eventually turn purple and form a rusty brown precipitate. Filter out the precipitate and store the resulting solution at 4 °C for future use.




2. Revised TAP medium

   Note: Reagents used in the revised TAP medium must be trace element grade. Reagents purchased from MACKLIN are only used in revised TAP.

   Reagent	Final concentration	Quantity or Volume
   Beijerinck’s solution	1% (v/v)	10 mL
   Phosphate solution	0.833% (v/v)	8.33 mL
   Tris-Acetate stock solution	1% (v/v)	10 mL
   Na2EDTA stock solution	25 µM	1 mL
   (NH4)6Mo7O24 stock solution	28.5 nM	1 mL
   Na2SeO3 stock solution	0.1 µM	1 mL
   ZnEDTA stock solution	2.5 µM	1 mL
   MnEDTA stock solution	6 µM	1 mL
   FeEDTA stock solution	20 µM	1 mL
   CuEDTA stock solution	2 µM	1 mL
   Milli-Q Water		Dilute with H2O to a final volume of 1 L
   Total		1 L

   For iron-limited TAP medium, add 0.01 mL of FeEDTA stock solution. For iron-overloading TAP medium, add 10 mL FeEDTA stock solution.

   
   

   Stocks for revised TAP medium:

   1. Beijerinck’s solutions (100×)

      Note: Store at 4 °C.

      Reagent	Final concentration	Quantity or Volume
      NH4Cl	750 mM	40 g
      CaCl2·2H2O	34 mM	5 g
      MgSO4·7H2O	40 mM	10 g
      Milli-Q Water		Dilute with H2O to a final volume of 1 L
      Total		1 L

   
   

   2. Phosphate solution (120×)

      Note: Store at 4 °C.

      Reagent	Final concentration	Quantity or Volume
      K2HPO4	82 mM	14.34 g
      KH2PO4	54 mM	7.26 g
      Milli-Q Water		Dilute with H2O to a final volume of 1 L
      Total		1 L

   
   

   3. Tris-Acetate stock solution (100×)

      Note: Store at 4 °C.

      Reagent	Final concentration	Quantity or Volume
      Tris base	2 M	242 g
      Acetic acid	1.7 M	100 mL
      Milli-Q Water		Dilute with H2O to a final volume of 1 L
      Total		1 L

   
   

   4. Na₂EDTA concentrate (Pre 1)

      Note: Store at 4 °C.

      Reagent	Final concentration	Quantity or Volume
      Na2EDTA·2H2O	125 mM	13.959 g
      Milli-Q Water		Dilute with H2O to a final volume of 300 mL
      Total		300 mL

      Dissolve 13.959 g of Na₂EDTA·2H₂O in about 250 mL of H₂O and titrate to pH 8.0 with trace element grade KOH (about 1.7 g).

   
   

   5. (NH₄)₆Mo₇O₂₄ concentrate (Pre 2)

      Note: Store at 4 °C.

      Reagent	Final concentration	Quantity or Volume
      (NH4)6Mo7O24·4H2O	285 µM	0.088 g
      Milli-Q Water		Dilute with H2O to a final volume of 250 mL
      Total		250 mL

   
   

   6. Na₂SeO₃ concentrate (Pre 3)

      Note: Store at 4 °C.

      Reagent	Final concentration	Quantity or Volume
      Na2SeO3	1 mM	0.043 g
      Milli-Q Water		Dilute with H2O to a final volume of 250 mL
      Total		250 mL

   
   

   7. Individual stock solutions (1,000×) of trace elements

      Note: Store at 4 °C.

      i. Na₂EDTA stock solution

      Reagent	Final concentration	Quantity or Volume
      Na2EDTA	25 mM	50 mL of Pre 1
      Milli-Q Water		200 mL
      Total		250 mL

      
      

      ii. (NH₄)₆Mo₇O₂₄ stock solution

      Reagent	Final concentration	Quantity or Volume
      (NH4)6Mo7O24	28.5 µM	25 mL of Pre 2
      Milli-Q Water		225 mL
      Total		250 mL

      
      

      iii. Na₂SeO₃ stock solution

      Reagent	Final concentration	Quantity or Volume
      Na2SeO3	0.1 mM	25 mL of Pre 3
      Milli-Q Water		225 mL
      Total		250 mL

      
      

      iv. ZnEDTA stock solution

      Reagent	Final concentration	Quantity or Volume
      ZnSO4·7H2O	2.5 mM	0.18 g
      Na2EDTA	2.75 mM	5.5 mL of Pre1
      Milli-Q Water		Dilute with H2O to a final volume of 250 mL
      Total		250 mL

      
      

      v. MnEDTA stock solution

      Reagent	Final concentration	Quantity or Volume
      MnCl2·4H2O	6 mM	0.297 g
      Na2EDTA	6 mM	12 mL of Pre1
      Milli-Q Water		Dilute with H2O to a final volume of 250 mL
      Total		250 mL

      
      

      vi. FeEDTA stock solution

      Reagent	Final concentration	Quantity or Volume
      FeCl3·6H2O	20 mM	1.35 g
      Na2EDTA	22 mM	2.05 g
      Na2CO3	22 mM	0.58 g
      Milli-Q Water		Dilute with H2O to a final volume of 250 mL
      Total		250 mL

      Note: Mix Na₂EDTA with Na₂CO₃ in Milli-Q water. Add FeCl₃·6H₂O after the first two components are dissolved. Do not use Pre 1.

      
      

      vii. CuEDTA stock solution

      Reagent	Final concentration	Quantity or Volume
      CuCl2·2H2O	2 mM	0.085 g
      Na2EDTA	2 mM	4 mL of Pre1
      Milli-Q Water		Dilute with H2O to a final volume of 250 mL
      Total		250 mL




3. Isolation buffer (IB buffer)

   Note: Store at 4 °C.

   Reagent	Final concentration	Quantity or Volume
   1 M Tris-HCl	20 mM	4 mL
   0.5 M EGTA	5 mM	2 mL
   0.5 M KCl	5 mM	2 mL
   0.2 M MgCl2	2 mM	2 mL
   Sucrose	6% (W/V)	12 g
   Milli-Q Water		Dilute with H2O to a final volume of 200 mL
   Total		200 mL

   
   

   Stocks for IB buffer:

   1. 1 M Tris-Cl

      Reagent	Final concentration	Quantity or Volume
      Tris base	1 M	12.114 g
      Milli-Q Water		Dilute with H2O to a final volume of 100 mL
      Total		100 mL

      Dissolve 12.114 g of Tris base in 50 mL of H₂O, adjust the pH to 7.6 with HCl, and adjust to 100 mL.

   
   

   2. 0.5 M EGTA

      Reagent	Final concentration	Quantity or Volume
      EGTA	0.5 M	19 g
      Milli-Q Water		Dilute with H2O to a final volume of 100 mL
      Total		100 mL

   
   

   3. 0.5 M KCl

      Reagent	Final concentration	Quantity or Volume
      KCl	0.5 M	3.725 g
      Milli-Q Water		Dilute with H2O to a final volume of 100 mL
      Total		100 mL

   
   

   4. 0.2 M MgCl₂

      Reagent	Final concentration	Quantity or Volume
      MgCl2	0.2 M	1.9 g
      Milli-Q Water		Dilute with H2O to a final volume of 100 mL
      Total		100 mL




4. OptiPrep gradient

   Note: OptiPrep density gradient medium needs to be dried into iodixanol powder at 50 °C before use. Store at 4 °C.

   Reagent	Final concentration	Quantity or Volume
   OptiPrep density gradient	80% (w/v)	3.2 g of iodixanol powder in 4 mL of IB buffer
   	60% (w/v)	2.4 g of iodixanol powder in 4 mL of IB buffer
   	40% (w/v)	1.6 g of iodixanol powder in 4 mL of IB buffer
   	37% (w/v)	1.48 g of iodixanol powder in 4 mL of IB buffer
   	34% (w/v)	1.36 g of iodixanol powder in 4 mL of IB buffer
   	27% (w/v)	1.08 g of iodixanol powder in 4 mL of IB buffer
   	24% (w/v)	0.96 g of iodixanol powder in 4 mL of IB buffer
   	15% (w/v)	0.6 g of iodixanol powder in 4 mL of IB buffer




5. 2 M DTT

   Note: Store at -20 °C.

   Reagent	Final concentration	Quantity or Volume
   DTT	2 M	0.3085 g
   Milli-Q Water		Dilute with H2O to a final volume of 1 mL
   Total		1 mL

Laboratory supplies

1. Blue cap bottles, 1 L (Beyotime, catalog number: FBT008), 500 mL (Beyotime, catalog number: FBT006), 250 mL (Beyotime, catalog number: FBT002)

2. 1 L beaker, 500 mL beaker, 250 mL beaker

3. 500 mL glass conical flask

4. Volumetric flasks of different volumes

5. 50 mL centrifuge tube

6. BD Precision Glide needle 25G×5/8 (Becton Dickinson Medical, catalog number: 301805)

7. Open-top thin wall ultra-clear tube, 5 mL (Beckman Coulter, catalog number: 344057)

8. 1.5 mL Eppendorf tubes

Equipment

1. Avanti^® J-E centrifuge (Beckman Coulter, model: 369001)

2. 96 mm diameter polypropylene conical bottle adapter (Beckman Coulter, model: 392078)

3. JS-5.3 AllSpin swinging-bucket rotor and buckets (Beckman Coulter, model: 368690)

4. JA-20 fixed-angle aluminum rotor (Beckman Coulter, model: 334831)

5. Desktop micro speed freezing centrifuge Optima MAX-XP (Beckman Coulter, model: 393315)

6. MLS-50 swinging-bucket rotor (Beckman Coulter, model: 367280)

7. Microcentrifuge (Eppendorf, model: 5424R)

Procedure

1. Cell cultures

   1. Inoculate cell wall–deficient strain cw15 cells grown on a TAP plate into two flasks containing 300 mL of TAP medium. Culture the cells at 24 °C with shaking (180 rpm) under continuous illumination (25 E·m^-2·s^-1) for 6 days. These cells can then be used to purify sta-LROs.

   2. For the iron overloading experiment, inoculate 5–6 mL of 2-day-old cw15 cells, cultured in revised TAP medium, into two flasks containing 300 mL of iron-limited TAP medium (0.2 µM Fe). Culture the cells under continuous light for 5 days with shaking (180 rpm) until the cell density reaches 3–5 × 10⁶ cells mL⁻¹. Subsequently, culture cells for another 26–28 h in iron-overloading TAP medium (200 µM Fe). These cells can then be used to isolate Fe-LROs.

      Note: It is recommended to purify LROs from freshly cultured cells. Storing cell pellets at -80 °C is not advised, as freeze-thaw cycles can cause partial cell lysis and chloroplast disruption, leading to impure LROs extraction.




2. Cell disruption

   1. Collect the cells from each flask (600 mL in total) by centrifugation at 3,500× g for 3 min.

   2. Discard the supernatant and wash the pellets once with 100 mL of IB buffer by resuspending with a pipette.

   3. Discard the supernatant again and resuspend the total pellets in 15 mL of IB buffer supplemented with a complete protease inhibitor cocktail and 2 mM DTT.

   4. Disrupt the cells by squeezing them through a syringe with a 25 G needle three times, ensuring most cells are opened without disrupting the chloroplast structure. During the squeeze process, ensure that the cell suspension flows out of the syringe needle in a continuous stream rather than intermittently dripping.




3. First OptiPrep gradient ultracentrifugation

   1. Adjust the volume of each sample to 70 mL with IB buffer.

   2. Centrifuge the samples at 10,000× g for 10 min at 4 °C.

   3. Discard the supernatant and resuspend the pellets in 4 mL of 34% OptiPrep (add 8 μL of DTT and 40 μL of protein inhibitor cocktail before use).

   4. Prepare a discontinuous OptiPrep gradient with 1 mL each of 15%, 24%, 34% (with samples), 37%, and 40% OptiPrep. It is recommended to prepare the OptiPrep gradient solution fresh for each use. The gradient should be constructed from the bottom up, starting with the highest density gradient at the bottom of the ultracentrifuge tube. Using a 1 mL pipette, carefully layer each subsequent gradient along the inner wall of the centrifuge tube at the slowest possible speed. During the gradient preparation process, distinct boundaries between different density layers can be clearly observed when held up to the light. There is no need for pause time between the different density layers.

   5. Centrifuge the gradient at 50,000× g in a Beckman MLS-50 rotor for 60 min at 4 °C, setting the acceleration to the maximum level and the deceleration to coast. The pellet below the 40% layer (P1) is the purified total LROs. Purified LROs can be stored at -80 °C for subsequent protein identification and related experiments, with a recommended storage duration of no more than three months. For localization experiments such as staining or immunofluorescence, it is recommended to use the purified LROs immediately after preparation.

      Note: The above steps are applicable for the purification of both sta-LROs and Fe-LROs from cw15 cells.




4. Second OptiPrep gradient ultracentrifugation

   1. Discard the supernatant and resuspend the total sta-LROs with 2 mL of 40% OptiPrep (add 4 μL of DTT and 20 μL of protein inhibitor cocktail before use).

   2. Prepare a discontinuous OptiPrep gradient with 1 mL each of IB buffer, 27%, 40% (with samples), 60%, and 80% OptiPrep.

   3. Centrifuge the gradient at 50,000× g in a Beckman MLS-50 rotor for 60 min at 4 °C, setting the acceleration to the maximum level and the deceleration to coast.

   4. Collect each layer (L1: layer between 40% and 60%, L2: layer between 60% and 80%) and the pellet below 80% (P2) in 1.5 mL Eppendorf tubes, fill to 1.5 mL with IB buffer, and mix by repeated pipetting.

   5. Centrifuge at 15,000× g for 10 min at 4 °C.

   6. Discard the supernatant; pellets are subgroups of sta-LROs separated by different densities.

      Note: The second OptiPrep gradient ultracentrifugation is used to separate total sta-LROs into subgroups, as most Fe-LROs accumulate in the pellet below 80% OptiPrep due to their high density.

Validation of protocol

This protocol has been used and validated in the following research article:

1. Long et al. [15]. Structural and functional regulation of Chlamydomonas lysosome-related organelles during environmental changes. Plant Physiology. DOI: 10.1093/plphys/kiad189

General notes and troubleshooting

General notes

1. Set the acceleration to the maximum level and the deceleration to coast during ultracentrifugation.

2. Vacuum must be applied prior to ultracentrifugation.

Acknowledgments

This study was supported by the National Key R&D Program of China (2020YFA0907400). This protocol was described and validated in the following research article: Long et al. [15]. Structural and functional regulation of Chlamydomonas lysosome-related organelles during environmental changes. Plant Physiology. DOI: 10.1093/plphys/kiad189.

Competing interests

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as potential conflicts of interest.

References

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