Materials and reagents

Reagents

1. Linseed oil OLEOS (Russia) (unrefined cold pressed linseed oil, refractive index n20/D 1.4795, density 0.93 g/mL at 25 °C), https://oleos-info.ru/product/lnyanoe-maslo/

2. Whey protein isolate (WPI) California Gold Nutrition (USA), https://www.californiagoldnutrition.com/products/california-gold-nutrition-sport-whey-protein-isolate-unflavored-5-lb-2-27-kg-76479 (27 g protein, 6.2 g BCAAs, 4.7 g glutamic acid, low lactose. Lactose is used as an excipient, which is not involved in the formation of emulsion microgels. No additives or flavor enhancers)

3. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417)

4. Tetramethylrhodamine isothiocyanate (TRITC) (Sigma-Aldrich, catalog number: T0820)

5. Fluorescein isothiocyanate isomer I (FITC) (Sigma-Aldrich, catalog number: F7250)

6. Rhodamine B isothiocyanate (RITC) (Sigma-Aldrich, catalog number: 283924)

7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888)

8. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911)

9. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C4901)

10. Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: S6014)

11. Dimethyl sulfoxide (DMSO) (EcoChemAnalyt, Russia, CAS: 67-68-5)

12. Urea (CH₄N₂O) (ReaChem, Russia, State Standard 2081-92, CAS: 57-13-6)

13. Ammonium chloride (NH₄Cl) (ReaChem, Russia, State Standard 3773-72, CAS: 12125-02-9)

14. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (ReaChem, Russia, State Standard 4523-77, CAS: 10034-99-8)

15. Sodium sulfate (Na₂SO₄) (ReaChem, Russia, State Standard 4166-76, CAS: 7757-82-6)

16. Sodium dihydrogen phosphate dihydrate (NaH₂PO₄·2H₂O) (ReaChem, Russia, State Standard 245-76, CAS: 13472-35-0)

17. Sodium hydrogen phosphate (Na₂HPO₄) (ReaChem, Russia, State Standard 11773-76, CAS: 7558-79-4)

18. Hydrochloric acid (HCl) (EcoChemAnalyt, Russia, State Standard 14261-77, CAS: 7647-01-0)

19. Sodium hydroxide (NaOH) (EcoChemAnalyt, Russia, State Standard 4328-77, CAS: 1310-73-2)

20. Trypsin (Sigma-Aldrich, catalog number: T4799)

21. Milli-Q water (Merck Millipore, model: Milli-Q^TM Advantage A10^TM, Germany)

    Note: It is possible to use linseed oil from another manufacturer. However, it is worth paying attention to its properties (the refractive index n20/D of 1.4795 and the density of 0.93 g/mL at 25 °C).

Solutions

1. Saline 0.9% NaCl (w/v) solution (see Recipes)

2. 2.5% WPI solution (see Recipes)

3. 5% WPI solution (see Recipes)

4. 7.5% WPI solution (see Recipes)

5. 5 mg/mL FITC solution (see Recipes)

6. 1 M NaOH solution (see Recipes)

7. PBS buffer (0.1 M, pH 8.3) (see Recipes)

8. Artificial urine solution (see Recipes)

9. 0.5 mg/mL trypsin in Tris-HCl buffer solution (20 mL) (see Recipes)

10. 70% ethanol (see Recipes)

Recipes

Note: In all recipes, liquid is added to the dry sample until the final specified volume is reached (“total” volume) in order to obtain an accurate solution concentration. For example, in Recipe 1, H₂O is added to the dry sample of NaCl until the final volume of 1 L is reached (instead of just adding 1 L of H₂O to the dry sample of NaCl). The volume of liquid is indicated in tables as a necessary recommended volume (not as a precise volume).

1. Saline 0.9% NaCl (w/v) solution

   *w/v: weight (g) per volume (100 mL).

   Reagent	Final concentration	Amount
   NaCl	0.9 % (w/v)	9 g
   H2O	n/a	1 L
   Total	n/a	1 L




2. 2.5% WPI (w/v) aqueous solution

   Reagent	Final concentration	Amount
   WPI	2.5 %	250 mg
   Saline 0.9% NaCl solution	0.9% (w/v)	10 mL
   Total	n/a	10 mL

   Caution: This solution should be mixed thoroughly. Before use, please wait until the foam at the surface of the solution disappears.

   Note: For the FITC conjugation procedure, WPI solution is prepared as described above in Recipe 2, but instead of water the PBS (pH 8.3) is used for protein dissolution.




3. 5% WPI (w/v) aqueous solution

   Reagent	Final concentration	Amount
   WPI	5%	500 mg
   Saline 0.9% NaCl solution	0.9% (w/v)	10 mL
   Total	n/a	10 mL

   Caution: This solution should be mixed thoroughly. Before use, please wait until the foam at the surface of the solution disappears.

   Note: For the FITC conjugation procedure, WPI solution is prepared as described above in Recipe 3, but instead of water the PBS (pH 8.3) is used for protein dissolution.




4. 7.5% WPI (w/v) aqueous solution

   Reagent	Final concentration	Amount
   WPI	7.5%	750 mg
   Saline 0.9% NaCl solution	0.9% (w/v)	10 mL
   Total	n/a	10 mL

   Caution: This solution should be mixed thoroughly. Before use, please wait until the foam at the surface of the solution disappears.

   Note: For the FITC conjugation procedure, WPI solution is prepared as described above in Recipe 4, but instead of water the PBS (pH 8.3) is used for protein dissolution.




5. 5 mg/mL FITC solution

   Reagent	Final concentration	Amount
   FITC	5 mg/mL	5 mg
   DMSO	n/a	1 mL
   Total	n/a	1 mL

   Note: Since a 5 mg sample is difficult to weigh, it is recommended to prepare a larger weight of FITC, which will require a larger volume of DMSO to prepare the final solution with the given FITC concentration of 5 mg/mL. For this, you need to proportionally increase FITC mass and DMSO volume. For example, weigh out 25 mg of FITC, then add DMSO until the volume reaches 5 mL. In this way, you will obtain 5 mL of a final FITC solution of 5 mg/mL.




6. 1 M NaOH solution (100 mL)

   Reagent	Final concentration	Amount
   NaOH	1 M	4 g
   H2O	n/a	100 mL
   Total	n/a	100 mL




7. PBS solution (0.1 M, pH = 8.3)

   Reagent	Final concentration	Amount
   PBS	1×	1 tablet
   H2O	n/a	200 mL
   Total	n/a	200 mL

   Note: Adjust pH of the resulting solution to 8.3 using 1 M NaOH and 1 M HCl solutions.




8. Artificial urine solution (pH 6.2)

   Reagent	Final concentration	Amount
   Urea	n/a	24.27 g
   NaCl	n/a	6.34 g
   KCl	n/a	4.50 g
   NH4Cl	n/a	1.61 g
   CaCl2	n/a	0.67 g
   MgSO4·7H2O	n/a	1.0 g
   NaHCO3	n/a	0.34 g
   Na2SO4	n/a	0.26 g
   NaH2PO4·H2O	n/a	1.0 g
   Na2HPO4	n/a	0.11 g
   H2O	n/a	2 L
   Total	n/a	2 L

   Note: The dry weights and the liquid volume can be changed proportionally to each other depending on needs (for example, for 1 L, the masses of dry samples are correspondingly reduced by two times relative to the masses given in the table). It is recommended to add weights in the order of priority shown in the table. The solution should be stirred for 3 h at 22 °C. The prepared artificial urine solution should be stored at 2–8 .




9. 0.5 mg/mL trypsin in Tris-HCl buffer solution (20 mL)

   Reagent	Final concentration	Amount
   Trypsin	0.5 mg/mL	10 mg
   Tris base	1 M	2.42 g
   H2O	n/a	20 mL
   Total	n/a	20 mL

   Note: Adjust pH of the Tris solution to 7.5 using HCl 1 M. The solution should be stored at 2–8 °C.




10. Ethanol 70%

    Reagent	Final concentration	Amount
    Ethanol 96%	70%	1 L
    H2O	n/a	371 mL
    Total	n/a	1,371 mL

    Note: When mixing these liquids, you must strictly add ethanol to water, not vice versa.

Laboratory supplies

1. Centrifuge tubes with flat cap, 15 mL (JetBioFil, catalog number: CFT550150)

2. Centrifuge tubes with flat cap, 50 mL (JetBioFil, catalog number: CFT500500)

3. Microcentrifuge tubes, 1.5 mL (JetBioFil, catalog number: CFT000015)

4. Microcentrifuge tubes, 2.0 mL (JetBioFil, catalog number: CFT000020)

5. Laboratory glass jar 100 mL, with divisions, with screw lid, dark glass (MiniMed, catalog number: 10007205)

6. Vial 2 mL, dark glass (ALWSCI Technologies, catalog number: C0001177)

7. Vial 10 mL, clear glass, 22 mm × 52 mm (ALWSCI Technologies, catalog number: C0000053)

8. Dialysis bag M-Cel, pore diameter 14 kDa (Viscase, catalog number: 2141-1425)

9. Dialysis bag clamp (Scienova GmbH, catalog number: 40329)

10. Laboratory beaker, clear glass, 2 L (MiniMed, catalog number:10003807)

11. Cellulose acetate membrane filter, 0.45 μm (Sartorius, catalog number: 11106-37-N)

12. Membrane filters PP, 10 μm (Gluvex, catalog number: MLPP1001000)

13. Glass for microslides (MiniMed, catalog number: 12003421)

14. Cover glass for microslides (MiniMed, catalog number: 12003309)

15. Syringe 1 mL (BD Micro-Fine Plus, catalog number: 320935)

16. 96-well V-bottom plate, conical bottom, non-treated, no lid (Costar, catalog number: 3897)

17. Pipette microtips, 2–20 μL (JetBioFil, catalog number: PPT100020)

18. Pipette microtips, 10–200 μL (JetBioFil, catalog number: PPT000200)

19. Pipette microtips, 100–1,000 μL (JetBioFil, catalog number: PPT000000)

20. Petri dishes, D 9.0 cm (JetBioFil, catalog number: MCD000090)

21. Adson microsurgical tweezers, 130 mm (Medical Equipment, catalog number: MF-2000)

22. Tissue tweezers, 130 mm (Medical Equipment, catalog number: MF-2102)

Equipment

1. Ultrasonic homogenization and Bandelin Sonopuls HD 2070 homogenizer at a frequency of 20 kHz and a power density of 1 W/cm² (Germany)

2. Amicon^® Stirred Cell 50 mL (Merck Millipore)

3. Magnetic stirrer (IKA)

4. Magnetic stirring bar (IKA, model: IKAFLON^® 15)

5. Multifunctional refrigerated centrifuge (Eppendorf, model: 5810R)

6. Microplate reader (BMG Labtech, model: CLARIO Star Plus)

7. Drybath thermo shaker (Thermo Scientific)

8. Inverted microscope with a 40× objective (Olympus IX73)

9. Water purification system for ultrapure water (Merck Millipore, model: Milli-Q^TM Advantage A10^TM)

10. Single-channel variable volume dispenser 100–1,000 mL, 10–100 mL, 20–200 mL, 5–50 mL, and 1,000–5,000 mL (Thermo Fisher Scientific)

Software and datasets

1. ClarioStar MARS 4.01 R2 (BMG LabTech, Germany)

2. ImageJ software 1.51j8 (National Institutes of Health, USA) (the open-source version is available online for free for download)

3. OriginPro 2018 SR1 (OriginLab Corporation, USA)

4. Excel 2019 (Microsoft Cooperation, USA)

Procedure