Step-by-Step Protocol for Fluorescence-Based Analysis of Uptake in Transporter-Expressing Xenopus laevis Oocytes

Materials and reagents

Biological materials

1. Xenopus laevis oocytes stage V–VI. Ovary lobes were kindly provided by Prof. Stephan Pless at the Department of Drug Design and Pharmacology, University of Copenhagen. Ovarian lobes were obtained shortly after surgical removal and transported to the laboratory in storage buffer supplemented with antibiotics. Transport of ovarian lobes to our lab was performed at ambient temperature and typically required 15–20 min, and collagenase treatment was commenced shortly after arrival. Defolliculated oocytes can be obtained commercially from Ecocyte Bioscience, Xenoocyte, or the European Xenopus Resource Centre (EXRC). Defolliculation and microinjection of stage V–VI oocytes followed the general procedures described in previous protocols [26–31].

Reagents

1. Esculin (Merck, Millipore, CAS: 66778-17-4, microbiology grade)

2. Sodium chloride (NaCl) (CAS: 7647-14-5, molecular biology grade)

3. Calcium chloride (CaCl₂) (CAS: 10035-04-8, molecular biology grade)

4. Magnesium chloride (MgCl₂) (CAS: 7791-18-6, molecular biology grade)

5. Potassium chloride (KCl) (CAS: 7447-40-7, molecular biology grade)

6. HEPES (CAS: 7365-45-9, molecular biology grade)

7. MES (CAS: 1266615-59-1, molecular biology grade)

8. DMSO (CAS: 67-68-5, ≥99.7%, Hybri-Max^TM grade); prepare 10% (v/v) DMSO and store at room temperature in a sealed container

9. Methanol (CAS: 67-56-1, ≥99.9%, HPLC grade); prepare 20% (v/v) methanol and store at room temperature in a sealed container

10. Ethanol (CAS: 64-17-5, 96% vol, molecular biology grade); prepare 70% (v/v) ethanol by dilution of 96% ethanol without correction; store at room temperature in a sealed container

11. MilliQ water

12. Amikacin disulfate salt (Thermo Scientific Chemicals, CAS no. 39831-55-5, catalog number: 455190050)

13. mMessage mMachine T7 Transcription kit (InVitrogen, Thermo Fisher Scientific, catalog number: AM1344)

14. Oocyte expression vector (e.g., pNB1 [32]) with gene of interest (SUC1 is used in the Validation of protocol section)

Solutions

1. Kulori buffer, pH 7.4 (see Recipes)

2. Kulori buffer, ph 4–6.5 (see Recipes)

3. 50 mM esculin in DMSO (see Recipes)

Recipes

1. Kulori buffer, pH 7.4

2. Kulori buffer, pH 4–6.5

Storage: Kulori solutions can be prepared in advance and stored at 4 °C. Under these conditions, the buffer is typically stable for several weeks, provided that no precipitation, turbidity, or microbial growth is observed. To minimize degradation of antibiotics, it is recommended to add antibiotics only to the volume required for immediate use rather than to the entire stock solution. Alternatively, a 10× Kulori stock can be prepared by increasing the concentration of all components tenfold and storing in 50-mL aliquots at -20 °C for several months. Prior to use, the stock should be diluted 1:10 with MilliQ water, and the pH verified and adjusted if necessary.

3. 50 mM esculin in DMSO

Esculin stock can be prepared in advance and stored at -20 °C. In our laboratory, esculin stocks stored under these conditions have been used successfully after at least 6 months of storage. To minimize repeated freeze/thaw cycles, we suggest preparing stock aliquots.

Laboratory supplies

1. Oocyte pipette (Pasteur pipettes with tips cut and smoothed) or plastic pipettes (e.g., BRAND^TM, catalog number: 15202699 with Fisherbrand^TM Dropper Bulb, catalog number: 03-448-26; alternative: disposable plastic pipettes, e.g., Pastette, catalog number: LW4111)

2. Petri dishes, non-treated, 55 mm in diameter (any standard laboratory grade)

3. Petri dishes, non-treated, 90 mm in diameter (any standard laboratory grade)

4. 48-well plate (Sarstedt, catalog number: 83.3923 is used in this protocol, but any standard laboratory grade should work)

5. 1.5 mL microcentrifuge tubes (any standard laboratory grade)

6. P1000 pipette tips (any standard laboratory grade)

7. P200 pipette tips (any standard laboratory grade)

8. P10 pipette tips (any standard laboratory grade)

9. Ice

10. Black 96-well plate (Thermo Scientific^TM, catalog number: 237108)

Equipment

1. Plate reader (BioTek, model: Synergy H1 Microplate reader)

2. Nanoject II (Drummond Scientific Company, Broomall, PA, USA)

3. Centrifuge for microcentrifuge tubes (HERMLE Labortechnic GmbH, Germany, model: Z216MK, and Thermo Scientific HERAEUS, Fresco 21, catalog number: 10651805; however, any centrifuge with a speed of >19,000× g can be used)

Procedure