A Universal Resazurin-Based Viability Assay for Prokaryotic and Eukaryotic Cells in 2D and 3D Cultures

Materials and reagents

Biological materials

1. MDA-MB-231 cell line (ATCC, catalog number: CRM-HTB-26)

2. Staphylococcus aureus (ATCC, catalog number: 27543)

Reagents

1. Dulbecco’s modified Eagle medium high glucose (DMEM) (Sigma Merck, catalog number: D5648) or RMPI media (Sigma, Merck, catalog number: R8005)

2. Sodium bicarbonate (NaHCO₃) (J.T. Baker, catalog number: 3506-01)

3. L-glutamine (Gibco, catalog number: 20530-081)

4. Amphotericin B (Sigma-Aldrich, catalog number: A2942)

5. Fetal bovine serum (FBS) (BioWest, catalog number: BIO-S1400)

6. Bovine calf serum (BCS) (BioWest, catalog number: S0400-500)

7. Penicillin-streptomycin (Gibco, catalog number: 15140-122)

8. Trypsin (Sigma-Aldrich, catalog number: T4799-5G)

9. Ethylenediaminetetraacetic acid (EDTA) (J.T. Baker, catalog number: 8993-01)

10. Resazurin sodium salt (Sigma-Aldrich, catalog number: R7017)

11. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650)

12. Actinomycin D (Sigma-Aldrich, catalog number: A9415)

13. Gentamicin sulfate (SON’S, catalog number: 87882 SSA IV)

14. Trypticase soy broth (TSB) (BD Bioxon, catalog number: 211825)

15. Sodium chloride (NaCl) (J.T. Baker, catalog number: 3624-01)

16. Monobasic potassium phosphate (KH₂PO₄) crystal (J.T. Baker, catalog number: 3824-01)

17. Potassium chloride (KCl) (J.T. Baker, catalog number: 3040-01)

18. Disodium phosphate (Na₂HPO₄) (J.T Baker, catalog number: 3828-01)

19. Agarose (Invitrogen, catalog number: 16500-100)

20. MilliQ water, sterile (J.T. Baker, catalog number: 4220-20)

21. Hydrochloric acid (HCl) (J.T. Baker, catalog number: 9535-05)

22. Sodium hydroxide (NaOH) (Macron, fine chemicals, catalog number: 7708-10)

23. Absolute ethanol (J.T. Baker, catalog number: 9000-03)

24. Sodium bicarbonate (NaHCO₃) (J.T Baker, catalog number: 3506-01) 134

Solutions

1. Culture medium (complete DMEM medium) (see Recipes)

2. 1.5% agarose solution (see Recipes)

3. Trypticase soy broth (TSB) medium (see Recipes)

4. 1× PBS solution, pH 7.4 (see Recipes)

5. 0.04% Trypsin-EDTA (see Recipes)

6. 500 μM actinomycin D stock solution (see Recipes)

7. Gentamicin stock solution (see Recipes)

8. Resazurin stock solution (see Recipes)

9. 1 M HCl (see Recipes)

10. 1 M NaOH (see Recipes)

11. 70% Ethanol (see Recipes)

Recipes

1. Culture medium (complete DMEM medium)

a. Dissolve DMEM powder in sterile distilled water and stir until completely dissolved.

b. Add 3 g of NaHCO₃.

c. Adjust the pH to 7.4 by carefully adding drops of NaOH (1 M) or HCl (1 M), as needed.

d. Under a laminar flow hood, supplement the medium with the following components:

i. 5% FBS (50 mL)

ii. 5% BCS (50 mL)

iii. 1% penicillin-streptomycin (10 mL)

iv 0.05% amphotericin B (500 μL)

v. 0.05% L-glutamine 200 mM (500 μL)

e. Sterilize the media by filtration using a 0.22 mm MCE membrane in a vacuum filtration system.

Note: MDA-MB-231 cells are routinely maintained in this medium. Once prepared and sterilized, the medium can be stored in a refrigerator at 4 °C and used within 4 weeks.

2. 1.5% agarose solution

a. Add 1.5 g of agarose to 100 mL of triple-distilled water in a glass flask.

b. Sterilize the solution by autoclaving for 15 min.

Note: This agarose solution is used to generate non-adherent surfaces in standard 96-well plates. The prepared solution should be sterilized by autoclaving and used fresh or maintained at ~60–70 °C until dispensing.

3. TSB medium

a. Dissolve trypticase soy broth in approximately 800 mL of distilled water using a magnetic stirrer.

b. Transfer the solution to a 1 L volumetric flask and adjust the final volume to 1 L with distilled water.

c. Sterilize by autoclaving at 120 °C for 20–30 min.

d. Allow the solution to cool to room temperature before use. Store at 4 °C if not used immediately.

4. 1× PBS solution, pH 7.4

a. Dissolve all components in approximately 800 mL of distilled water using a magnetic stirrer until the solution is clear and no particulates remain.

b. Calibrate the pH meter using standard buffer solutions, then titrate the solution to pH 7.4 by adding 1 M HCl or 1 M NaOH dropwise with continuous stirring. Allow the reading to stabilize after each addition.

c. Transfer the solution to a 1 L volumetric flask and adjust the final volume to 1 L with distilled water. Mix thoroughly by inverting the flask several times.

d. Transfer the solution to an autoclave-safe bottle, loosen the cap slightly to allow pressure equilibration, and autoclave at 121 °C for 20–30 min.

e. After autoclaving, allow the solution to cool to room temperature before use. Store appropriately if not used immediately, and label with the solution name, pH, and preparation date.

Note: Alternatively, a commercial PBS solution may be used.

5. 0.04% Trypsin-EDTA

a. Prepare 1 L of sterile 1× PBS according to Recipe 4.

b. In a separate sterile container, dissolve 390 mg of trypsin and 85 mg of NaCl in 10 mL of the prepared 1× PBS. Mix gently until completely dissolved.

c. To the remaining PBS (~990 mL), add 370 mg of EDTA and stir until fully dissolved.

d. Combine the trypsin-NaCl solution with the EDTA-containing PBS and stir thoroughly to ensure complete mixing.

e. Under aseptic conditions, filter-sterilize the combined solution using a 0.22 μm vacuum filtration unit.

f. Aliquot the sterile solution into appropriate volumes (e.g., 50 mL), label clearly, and store at -20 °C until use.

Note: This trypsin-EDTA solution is used for cell detachment.

6. 500 μM actinomycin D stock solution

Caution: Actinomycin D is a potent cytotoxic and teratogenic agent. Wear appropriate personal protective equipment (PPE), including gloves and safety goggles, and handle all materials in a designated chemical fume hood or biological safety cabinet.

a. Working under aseptic conditions in a cell culture flow hood, dissolve the appropriate amount of actinomycin D in sterile DMSO to achieve a final concentration of 500 μM.

b. Mix gently by pipetting or vortexing until completely dissolved.

c. Dispense the solution into amber or foil-wrapped microcentrifuge tubes to protect from light.

d. Label each aliquot clearly with the compound name, concentration, and date of preparation.

e. Store protected from light at -20 °C until use. Avoid repeated freeze-thaw cycles.

Note: Actinomycin solution can be stored at -20 °C for up to one year.

7. Gentamicin stock solution (100 μg/mL)

Note: Common working stocks are 10 mg/mL or 50 mg/mL. Adjust the volume of water accordingly based on the mass of gentamicin powder used.

a. Dissolve the required mass of gentamicin sulfate powder in an appropriate volume of sterile distilled water to achieve the desired stock concentration (e.g., 10 mg/mL or 50 mg/mL). Mix gently until completely dissolved.

b. Under aseptic conditions in a cell culture flow hood, sterilize the solution by filtration through a 0.22 μm syringe filter.

c. Aliquot the sterile solution into appropriately sized, sterile microcentrifuge tubes or vials.

d. Label each aliquot with the antibiotic name, concentration, and date of preparation.

e. Store at 4 °C for short-term use (up to one month) or at -20 °C for long-term storage. Avoid repeated freeze-thaw cycles.

8. Resazurin stock solution (0.15% w/v)

a. Resazurin stock solution, 1 mg/mL

Note: This protocol yields a 1 mg/mL resazurin stock solution, commonly used as a cell viability indicator in proliferation and cytotoxicity assays. This concentration is typically used as a 10× or 100× stock depending on the assay format.

a. Accurately weigh the required mass of resazurin sodium salt using an analytical balance. For a 1 mg/mL stock, weigh 100 mg of resazurin sodium salt to prepare 100 mL of solution.

b. Transfer the powder to a sterile 100 mL volumetric flask and dissolve in approximately 80 mL of sterile 1× PBS.

c. Stir gently on a magnetic stirrer or swirl by hand until the powder is completely dissolved, yielding a uniform blue solution.

d. Adjust the final volume to 100 mL with sterile 1× PBS. Mix thoroughly by inverting several times.

e. Immediately cover the container with aluminum foil or use an amber bottle to protect the solution from light, as resazurin is photosensitive.

f. Under aseptic conditions in a cell culture flow hood, filter-sterilize the solution through a 0.22 μm membrane filtration unit.

g. Aliquot the sterile solution into light-protected tubes (amber microcentrifuge tubes or clear tubes wrapped in aluminum foil).

h. Label each aliquot with the solution name, concentration (1 mg/mL), and date of preparation.

i. Store protected from light at 4 °C until use.

Note: Resazurin stock solution can be stored at 4 °C for up to 6 months or longer at -20 °C.

b. Resazurin working solution (0.15 mg/mL) from the 1 mg/mL stock

Note: This protocol yields a 0.15 mg/mL (150 μg/mL) resazurin working solution, typically used as an intermediate dilution for cell viability assays. This concentration is often used as a 10× stock, diluted 2:10 in cell culture medium to achieve a working concentration of 30 μg/mL.

a. Calculate the required dilution factor using the formula C₁V₁ = C₂V₂, where:

C₁ = Initial concentration (1 mg/mL)

C₂ = Desired concentration (0.15 mg/mL)

V₂ = Desired final volume of working solution

V₁ = Volume of stock solution needed

Example calculation for 10 mL of working solution:

(1 mg/mL) × V₁ = (0.15 mg/mL) × (10 mL)

V₁ = (0.15 × 10)/1 = 1.5 mL of stock solution

b. Under aseptic conditions in a cell culture flow hood, aliquot the calculated volume of 1 mg/mL resazurin stock solution into a sterile container.

c. Add sterile 1× PBS (or sterile cell culture medium, depending on your application) to achieve the desired final volume. For 10 mL, add 1.5 mL of resazurin stock (1 mg/mL) + 8.5 mL of sterile 1× PBS.

d. Mix gently by pipetting or swirling until thoroughly combined.

e. Protect the solution from light by using an amber tube or by covering with aluminum foil.

f. If not used immediately, aliquot into light-protected tubes and label with the concentration (0.15 mg/mL) and date.

g. Store protected from light at 4 °C for short-term use (up to 2 weeks) or at -20 °C for longer storage. Avoid repeated freeze-thaw cycles.

9. 1 M HCl

a. Put on gloves and safety goggles. Work inside a fume hood.

b. Measure 9.05 mL of 37% HCl using a graduated cylinder or pipette.

c. Pour approximately 50–70 mL of MilliQ water into a 100 mL volumetric flask.

d. Slowly add the measured HCl into the water while gently swirling the flask.

e. Wait for the solution to cool down (it gets warm).

f. Add more MilliQ water until the bottom of the meniscus reaches the 100 mL mark.

g. Mix well by inverting the flask several times.

h. Transfer the 1 M HCl to a labeled glass bottle. Store at room temperature.

Caution: Always add acid to water, never water to acid.

10. 1 M NaOH

a. Put on gloves and safety goggles. Work in a well-ventilated area or fume hood.

b. Weigh 4 g of NaOH using a balance.

c. Pour approximately 50–70 mL of MilliQ water into a 100 mL volumetric flask or a glass beaker.

d. Slowly add the NaOH to the water while gently stirring with a glass rod.

Caution: Never add water to NaOH (the solution becomes very hot).

e. Wait for the solution to cool down to room temperature.

f. Transfer the solution to the volumetric flask (if using a beaker) and add more MilliQ water until the bottom of the meniscus reaches the 100 mL mark.

g. Mix well by inverting the flask several times.

h. Transfer the 1 M NaOH to a labeled plastic or glass bottle. Store at room temperature.

Caution: NaOH is caustic. Always add NaOH to water, not water to NaOH, to prevent violent boiling and splashing.

11. 70% ethanol

a. Measure 70 mL of absolute ethanol using a graduated cylinder.

b. Pour the ethanol into a 100 mL volumetric flask or a clean glass bottle.

c. Add MilliQ water slowly until the total volume reaches 100 mL.

d. Close the container and mix well by inverting or shaking gently.

e. Label the bottle as 70% ethanol with the date of preparation.

f. Store at room temperature.

Laboratory supplies

1. 96-well round-bottom assay plate (Corning, catalog number: 3797)

2. Non-adherent 96-well plates (see Recipes)

3. Cell culture plate, 96 wells, flat bottom, tissue culture (TC) grade (Nest, catalog number: 701001)

4. Cell culture dishes, 60 mm × 15 mm (NEST, catalog number: 705001)

5. Conical-bottom centrifuge tubes, 50 mL (Uniparts, catalog number: 32117F)

6. Conical-bottom centrifuge tubes, 15 mL (Uniparts, catalog number: 34117F)

7. Microcentrifuge tubes, 1.5 mL (Axygen, catalog number: MCT-150-C)

8. 1,000 μL pipette tips (Uniparts, catalog number: 51131)

9. 200 μL pipette tips (Uniparts, catalog number: 51121Y)

10. 10 μL pipette tips (Axygen, catalog number: T-300)

11. MF-Millipore 0.22 mm MCE membrane, 47 mm (Millipore, catalog number: GSWP04700)

12. Nitrile gloves (Kirkland Signature, catalog number: B0CJ4JMTQQ)

13. Serological pipettes 2 mL (Corning, catalog number: 4486)

14. Aluminum foil (Alupractuk, catalog number: ALU400)

Equipment

1. Centrifuge (PowerSpin^TM, model: C856)

2. Micropipettes

a. 0.5–10 μL single-channel pipettor (Axygen^® Axypet^®, catalog number: AP-10)

b. 10–100 μL single-channel pipettor (Axygen^® Axypet^®, catalog number: AP-100)

c. 100–1,000 μL single-channel pipettor (Axygen^® Axypet^®, catalog number: AP-1000)

3. pH meter (Apera, model: PH700)

4. Orbital shaker (Benchmark, model: BT302)

5. Digital stirring hot plate (Thermo Scientific, model: SP131015Q)

6. Inverted microscope (Carl Zeiss, model: 37081)

7. Laminar flow cabinet (Thermo Fisher Scientific, model: 1340)

8. Incubator for cell culture (Thermo Fisher Scientific, model: 3422)

9. Neubauer chamber (Mariendfeld, catalog number: 0610010)

10. Microwave oven (standard laboratory or domestic units, 600–1,000 W) for agarose melting

11. Vacuum filtration system (Nalgene, catalog number: 300-4050)

12. Incubator for bacterial culture (Thermo Fisher Scientific, model: SHKE6000)

13. Microplate reader Varioskan Flash (Thermo Scientific, model: N06354)

14. Autoclave (Felisa, model: FE-397)

Software and datasets

1. ImageJ (https://imagej.net/ij/download.html)

2. GraphPad Prism 9.1.1 (https://www.graphpad.com/features)

Procedure