Fluorescence Measurement and Calibration of Intracellular pH in Starfish Oocytes

Materials and Reagents

1. Glass capillary for a glass micropipette, Microcap, 50 μl (Drummond Scientific Company, catalog number: 1-000-0500)
   A micropipette was made using the puller PC-100 system, as per the manufacturer’s instructions (Narishige, https://products.narishige-group.com/group1/PC-100/pipette/english.html). The properties (length + outer/inner diameter) of the ideal capillaries are shown in Figure 2A. To control precisely the flow volume out of the glass micropipette, a constriction was made using a handmade loop of platinum wire. Briefly, the micropipette was passed through the loop of the platinum wire. Then, the micropipette (3-5 mm from the end) was heated until a constriction of 3.5-5.0 µm in diameter was formed via the application of an electric current to the loop (see also Hiramoto, 1974).
2. Cover glasses (Matsunami, 18 mm x 18 mm, thickness 0.12-0.17 mm)
3. Silicone oil (Shin-Etsu Chemical Co.Ltd. catalog number: KF-96-100CS ) (Figure 2C)
4. A dextran (10-kD) BCECF conjugate (Fisher Scientific, Invitrogen^TM, catalog number: D1878 ) (Figure 2B)
   
   
   Figure 2. Materials and equipment used for oocyte manipulation. A. The constriction made in a glass micropipette acts as a brake. B. BCECF solution in a glass capillary. C. BCECF solution in a glass capillary is set in the capillary holding chamber filled with silicone oil. The glass micropipette (A) filled with silicone oil is inserted into the BCECF solution; 20 pL BCECF is aspirated into the micropipette. D. The oocyte holder. E. The manipulation chamber. F. The oocyte holder for 1-MA treatment. G. The manipulation chamber for 1-MA treatment. The chamber has an opening that allows the removal of an ASW volume (25%) and its replacement by the same volume of ASW containing 1-MA. D-G. All measurements are represented in mm unless otherwise indicated.


5. 1-methyladenine (KANTO CHEMICAL CO. INC., catalog number: 20131-1A )
6. Potassium aspartate (Tokyo Chemical Industry Co., Ltd., catalog number: A0922 )
7. HEPES, 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (DOJINDO, catalog number: 340-01371 )
8. Pipes, Piperazine-1,4-bis (2-ethanesulfonic acid) (DOJINDO, catalog number: 347-02224 )
9. KOH (Wako Pure Chemical Industries, Ltd., catalog number: 168-21815)
10. NP-40 (Nonidet P-40. Nacalai Tesque, catalog number: 252-23 )
11. NaCl (FUJIFILM Wako Pure Chemical Corporation, catalog number: 191-01665 )
12. KCl (KANTO CHEMICAL CO. INC., catalog number: 32326-00 )
13. MgCl₂•7H₂O (Wako Pure Chemical Industries, Ltd., catalog number: 135-00165 )
14. MgSO₄•7H₂O (Wako Pure Chemical Industries, Ltd., catalog number: 131-00405 ) 
15. H₃BO₃ (Showa Chemical Co. Ltd., catalog number: 0214-6250 )
16. CH₃COONH₄ (Wako Pure Chemical Industries, Ltd., catalog number: 019-02835 )
17. CaCl₂•2H₂O (Wako Pure Chemical Industries, Ltd., catalog number: 031-00435 )
18. Calcium-free ASW (see Recipes)
19. ASW (see Recipes)
20. Modified ASW pH 6.8 (see Recipes)
21. Standard pH solutions at pH 6.40-6.75 or 6.80-7.40 (see Recipes)
22. BCECF solution (see Recipes
    

Equipment

1. CMOS camera (Hamamatsu Photonics K.K., ORCA-Flash2.8, model: C11440-10C )
2. ECLIPSE Ti-U fluorescent microscope (Nikon Instech)
3. 4× 0.20 NA CFI super Fluor lens (Nikon Instech)
4. Navi h pH meter (HORIBA, pH METER, F-52)
5. Screw-controlled microinjector (Narishige, model: IM-9B )
   

Software

1. HCImage U11158-02, 03 (Hamamatsu Photonics K.K., https://hcimage.com/)

Procedure

Figure 3. Flowchart representing the procedure overview (Procedures A to F)

1. Oocyte preparation (Video 1)
   
   
   
   Video 1. Oocyte Preparation. This video was made at Ochanomizu Univ. according to guidelines from the Ochanomizu Univ. on Animal Care and approved by the Animal Research Ethics Board of Ochanomizu University.
   
   Maintain starfish (Asterina pectinifera) in laboratory aquariums supplied with circulating seawater at 14 °C (Murabe et al., 2020).
   
   1. Isolate oocytes or ovaries from female animals and treat them with calcium-free artificial seawater to eliminate follicle cells.
   2. Maintain follicle-free oocytes in artificial seawater (ASW) until use.
      


2.  Microinjection of BCECF into oocytes (Video 2)
   
   
   
   Video 2. Microinjection technique
   
   
   1. Generate a glass micropipette having a constriction of a few micrometers in diameter to control the flow speed out of the micropipette (Hiramoto, 1974; see also Materials and Reagents 1 and Figure 2A).
   2. Fill the constricted micropipette with silicon oil and connect it to the screw-controlled microinjector ( IM-9B Narishige).
   3. Microscopically aspirate 20 pl BCECF solution into the micropipette (Figures 2A-2C).
   4. Microinject the BCECF solution into a starfish oocyte sandwiched between the two coverslips of an oocyte holder (Chiba et al., 1992) (Figures 2D and 2E).
   5. Incubate oocytes injected with BCECF in ASW for 1 h at 23 °C before measuring the BCECF fluorescence intensity or the pH_i.
      Note: Incubation for 1 h is required for diffusion of injected BCECF.
      
   
   
3. Image acquisition (sequence-imaging) of the oocytes injected with BCECF before and after 1-MA stimulation (Video 3)
   
   
   
   Video 3. pH_i measurement using BCFCF
   
   
   1. To evaluate the camera “noise”, acquire a dark image (no light is delivered to the CMOS camera controlled by the HCImage acquisition system, which is connected to a fluorescence microscope equipped with differential interference contrast (DIC) optics including a 4× 0.20 NA CFI super Fluor lens).
   2. Set oocytes pre-injected with BCECF (as shown in Procedure B) in a manipulation chamber for 1-MA treatment at 23 °C (Figures 2F and 2G).
   3. Acquire a DIC image of 8-15 oocytes (Figure 4A).
   4. Acquire a fluorescence image of oocytes to configure the region of interest (ROI) (Figure 4B): surround the cytoplasm excluding undiffused BCECF and the GV area in the oocyte image to define the ROI, using the HCImage Analysis software. Most of the injected BCECF is diffusely distributed throughout the cytoplasm 1 h after injection; however, a small, brighter fraction, is usually retained at the site of injection near an oil drop. Importantly, this brighter region should be excluded from the ROIs (Figures 4C and 4D).
      
      
      Figure 4. Oocytes’ images acquisition. A. A representative oocytes’ DIC image. B. A fluorescence image of oocytes injected with BCECF. C. The ROI is defined using a green line. The background area is also colored in green. D. An enlarged oocyte image showing the ROI. The GV area is excluded from the ROI since GVBD causes a rapid change in fluorescence intensity.
   
   
   5. Select an area without oocytes to get the background fluorescence (Figure 4C).
   6. Before treatment with 1-MA, acquire fluorescence microscopic images of BCECF-injected oocytes, excited every 10 s at 436 nm and 495 nm for more than 5 min. This excitation alternation is obtained from a Xenon lamp using a filter wheel under the computational control of the HCImage acquisition system. Then, the emitted light passes through a dichroic beam splitter at 505 nm and through a 510-560 nm emission filter, and finally through the CMOS camera and is recorded by the HCImage acquisition system.
   7. Pause the CMOS camera, and aspirate 25% of the volume of ASW and subsequently add 25% of the volume of ASW containing 2 µM 1-MA (final concentration: 0.5 µM).
   8. Immediately after addition of 1-MA, turn on (or resume) the CMOS camera to obtain fluorescence images of the oocyte for 2-30 min. Refer to the original study for more detailed information regarding the procedure and prototypical images (Hosoda et al., 2019; Figure S5F, left panel) (see also Video 3).
      
   
   
4. Calculation of the average fluorescence intensity ratios, using the recorded images of BCECF-injected oocytes and the HCImage analysis system
   
   1. Open an Image Display window showing the fluorescence microscopic image sequence of the oocyte. If oocytes move during recording, new ROIs should be defined according to the procedure in Step C4.
   2. Calculate the fluorescence intensity ratios between 436 nm and 495 nm excitation from 8-15 oocytes, using the HCImage Analysis software.
   3. Average the ROI ratios of 8-15 oocytes at each time point.
   4. After the experiments, convert the average ratios to pH_i, using the procedure outlined in Procedure F.
      
   
   
5. pH_i clamping with modified ASW (sodium-free ASW containing CH₃COONH₄) and calibration after injection of standard solutions with known pH values
   Note: For oocytes treated with modified ASW at pH 6.8, the pH_i is expected to be clamped at ~7.0, because the pH_i values become ~0.2 higher than the pH values of the modified ASW (Moriwaki et al., 2013; Hosoda et al., 2019).To calibrate accurately the clamped pH_i, inject different sets of oocytes with at least three types of pH-standard solutions. When the actual (or real) pH_i is higher than the known pH of the injected standard solution, the BCECF fluorescence intensity ratio will decrease after injection since the pH_i also decreases upon injection of the standard solution at a lower pH. Conversely, when the actual pH_i is lower than the known pH of the injected standard solution, the BCECF fluorescence intensity ratio will increase. Thereafter, the actual pH_i should be between the pH values of the two injected standard solutions. When the actual pH_i is equal to that of the injected solution, the intensity ratio does not change. Using this method, pH_i differences as low as 0.05 can be detected. In this section, taking as an example modified ASW at pH 6.8, the pH_i of unstimulated oocytes was clamped, and different sets of clamped oocytes were injected with different pH-standard solutions (pH 7.00, 7.05, 7.10) to estimate the values of clamped pH; readers can expect to see three different outcomes as shown in Figures 5A, 5B and 5C, that allowed the calibration of pH_i.
   
   
   1. Remove BCECF-injected oocytes from the oocyte holder (Figure 2D) after Procedure B, and recover them in 20 μl ASW, using an adjustable-volume micropipette. Then, transfer them immediately to 1 ml sodium-free ASW containing CH₃COONH₄ at pH 6.8 (modified ASW pH 6.8). Thereafter, gently agitate the oocytes for 15 s.
   2. Transfer the oocytes in 20 μl modified ASW at pH 6.8 to freshly modified ASW (1 ml) at pH 6.8, and gently agitate the oocytes for 15 s. Repeat this procedure 5 times to completely eliminate Na+ (with modified ASW at pH 6.8).
   3. Sandwich immature oocytes with two cover glasses using the oocyte holder (Figure 2D).
   4. Define the ROI of an oocyte and select an area without oocytes, as background fluorescence–Steps C4 and C5.
      Note: The procedures in Steps E1 to E4 should be completed within 20 min to obtain reproducible data.
   5. Record a fluorescence image sequence of an oocyte for ≥ 100 s.
   6. To inject the pH-standard solution, pause the CMOS camera.
   7. Inject oocytes with 2% of the volume per oocyte of a pH-standard solution at pH 7.00; of note modified ASW at pH 6.8 may clamp pH_i at ~7.0 (Moriwaki et al., 2013; Hosoda et al., 2019).
   8. Rapidly turn on the CMOS camera system to record the changes in the fluorescence intensity ratio for at least 20 s.
   9. Measure the average fluorescence intensities inside the ROI and calculate the ratio of the averaged intensities between the 436 nm and 495 nm excitation wavelengths, using the HCImage analysis software. A typical result is shown (Figure 5A). In this particular example, the fluorescence intensity ratio decreased upon injection of the pH-standard solution. Thus, the clamped pH_i is estimated to be higher than pH 7.00. Therefore, in the next step, a standard solution with a pH higher than 7.00 should be used, in a way to cause an increase in the fluorescence intensity ratio, and ultimately to determine the real pH_i.
       
      
      Figure 5. Estimation of the pH_i in oocytes treated with modified ASW at pH 6.8. A. The standard solution at pH 7.00 was injected between 100 s and 140 s. Clamped pH_i was estimated to be higher than pH 7.00 in this oocyte. B. The standard solution at pH 7.10 was injected between 125 s and 185. Clamped pH_i was estimated to be lower than pH 7.10. C. The pH-standard solution at pH 7.05 was injected between 125 s and 220 s. No change in the BCECF fluorescence intensity ratio was observed. Therefore, the clamped pH_i is estimated to be pH 7.05.
   
   
   10. After the procedure described in Steps E1-E6, inject the oocytes with 2% volume per oocyte of a standard solution at pH 7.10; because pH 7.00 decreased the fluorescence intensity ratio (Figure 5A), pH 7.10 is expected to increase this ratio.
   11. Turn on the CMOS camera system and calculate the fluorescence ratio for the standard solution at pH 7.10, as mentioned in Steps E7-E9.
   12. A typical result is shown (Figure 5B). As expected, the fluorescence intensity ratio increased upon injection of the pH-standard solution. Thus, the clamped pH_i is estimated to be lower than pH 7.10 and higher than pH 7.00. Therefore, to find out a standard solution with an adequate pH value causing no change of the fluorescence intensity ratio, standard solutions 7.00 ≤ pH ≤ 7.10 should be considered.
   13. After the procedure described in Steps E1-E6, inject the oocytes with 2% of the volume per oocyte of a standard solution at pH 7.05, and then turn on the CMOS camera system and calculate the respective fluorescence ratio, as mentioned in Steps E7-E9.
   14. A typical result is shown (Figure 5C). As expected, the fluorescence intensity ratio did not change upon injection of the pH-standard solution, indicating that the actual pH_i should be equal to that of the injected solution (= pH 7.05).
       


6. Calibration of pH_i in normal seawater
   Note: The procedure in F should be conducted on the same day when the procedure in C is performed.
   
   1. To conduct a two-point calibration, treat immature oocytes (n = 8-15) with modified ASW to clamp the pH_i at higher and lower values, and obtain the respective BCECF high- and low-intensity ratios. Usually, modified SW at pH 6.4 (pH_i ~6.6) and that at pH 7.3 (pH_i ~7.5) can be used. The actual pH_i of oocytes in modified ASW is estimated as described in Procedure E.
   2. Calculate the average values for each point.
   3. Plot fluorescence ratios against pH_i 6.6 and pH_i 7.5 to obtain a standard linear calibration graph/function. More detailed information regarding this procedure is already provided in the original study (Hosoda et al., 2019; Figure S5D and E). Convert the ratio data from procedure E to pH_i values, using the standard linear calibration graph/function. Again, refer to the procedure in the original study for more detailed information (Hosoda et al., 2019; Figure S5F)
      

Recipes

1. Calcium-free ASW
   476 mM NaCl
   10 mM KCl
   36 mM MgCl₂
   18 mM MgSO₄
   20 mM H₃BO_3, adjusted to pH 8.2
2. ASW
   462 mM NaCl
   10 mM KCl
   36 mM MgCl₂
   18mM MgSO₄
   10 mM CaCl₂
   20 mM H₃BO₃, adjusted to pH 8.2
3. Modified ASW pH 6.8
   480 mM choline chloride
   55 mM MgCl₂
   5 mM KCl
   10 mM Pipes
   10 mM HEPES
   20 mM CH₃COONH₄
   9.2 mM CaCl₂
   pH 6.8 is adjusted by KOH
4. Standard pH solutions at pH 6.40-6.75 or 6.80-7.40
   0.5 M Pipes for pH 6.40-6.75; pH is adjusted by KOH
   0.5 M HEPES for pH 6.80-7.40; pH is adjusted by KOH
5. BCECF solution
   2 mM dextran (10-kD) conjugate BCECF
   100 mM potassium aspartate
   10 mM HEPES
   0.05% NP-40, pH 7.2
   pH 7.2 is adjusted by KOH
   

   Note: The pH of the ASW, the modified ASW, and the standard solutions should be adjusted at 23 °C using a Navi h pH meter (HORIBA), which has a resolution of 0.001 pH units. All experiments associated with pHi measurement should be performed at 23 °C.

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant no. 17K07405), the Takeda Science Foundation, and the Cooperative Program provided by the Atmosphere and Ocean Research Institute, University of Tokyo. This protocol was adapted from Moriwaki et al., 2013 and Hosoda et al., 2019 (for pHi calibration).

Competing interests

The authors declare no competing financial interests.

Ethics

Experiments were conducted at Ochanomizu Univ. according to guidelines from the Ochanomizu Univ. on Animal Care and approved by the Animal Research Ethics Board of Ochanomizu University.

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