Evaluation of Toxicity with Brine Shrimp Assay

Materials and Reagents

Brine shrimp assay

1. Latex gloves (KCWW, Kimberly-Clark, catalog number: 57330 )

2. 24-well plate, sterile and tissue-treated (Corning, catalog number: 3526 )

3. Pipette tips

4. Brine shrimp eggs (Artemia salina) (were purchase from Ocean Nutrition) and the Pure Artemia Cysts are coming from the Great Salt Lake and therefore geographical variations are not affecting the assay

5. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )

6. Sea salt (Tropic Marin)

Equipment

1. 2 L separating funnel with 24/29 joint

2. Stereo microscope Stemi 2000 (ZEISS)

3. Air Pump (Mouse M-106, two outputs 4w)

4. Soft light source such as fluorescent lamps 7w

5. Incubator

   Note: Any incubator is fine if it maintains 25 °C to 37 °C, 50-60% humidity and 12 h day lighting/ 12 h dark for 24 h.

Software

1. Microsoft Office Excel

Procedure

1. 1 g cysts of Artemia salina are soaked in 500 ml natural fresh water for one hour in a 2 L separating funnel. The funnel should not be covered on top.

2. 17 g of sea salt are dissolved in the 500 ml natural freshwater water above.

3. Facilitate the funnel with good aeration using an air pump at room temperature and under continuous illumination for 48 h (Figure 1).

   
   

   
   Figure 1. The nauplii incubation apparatus

   
   

4. After 48 h hatching, the nauplii released from the egg shells.

5. Collect nauplii at the bright side of the funnel (near the light source) by using a micropipette.

6. Transfer the separated larvae above in a small beaker containing NaCl 0.9%.

7. Introduce an aliquot (0.5 ml) from a small beaker about 10 to 20 nauplii to each well of 24-well plate. The total volume of the well of 24-well plate will be 1ml NaCl 0.9%.

8. Test the metallodrug at three concentrations, with a range of, for example 0.05, 0.5 and 5 μΜ with three replicates per concentration. The IC₅₀ value of the metallodrug towards the cancerous cell lines should be in this range of the tested concentrations. When an antibacterial or a fungicidal compound is tested, then the tested concentration should be the MIC and 2× MIC, 4× MIC values. In the case of hydrogel materials, discs with a diameter of 9 mm were added to each well.

9. The final volume of each well is 1 ml with NaCl 0.9% and the presence of the metallodrug.

10. Maintain the plates at 25°C in an incubator.

11. Examine, after 24 h the brine shrimps, using a stereoscope.

12. Larvae were considered alive if they exhibit internal or external movement during 10 s of observation.

13. Repeat each experiment three times.

Note: During the decapsulation of Artemia cyst, continuous aeration, using an air pump, should be done for proper hatching of the embryo, at 25 °C with simultaneously continuous illumination. For the acceptability of the test, up to 10% of mortality was admitted in the control.

Data analysis

Representative data

Selected icons of hatching eggs, live and dead nauplii brine shrimps which were treated with an agent are shown in Figure 2.

1. Observed larvae, and the dead larvae are considered those that did not exhibit any internal or external movement in 10 s of observation.

2. Count numbers of dead larvae.

3. The (%) mortality of Artemia salina larvae was calculated according to Abbott 1987:

   
   

   M(% vs. control)=[(L_C−L_T)/L_C]×100

   
   

   where M is mortality; L_C, living nauplii in the control after 24 h; L_T, living nauplii with the tested agent after 24 h.

Acknowledgments

The protocol for Artemia salina assay was adapted and modified from a previous study (Syahmiet al., 2010; da Silveira Carvalho et al., 2017 ; Rahmanet al., 2018; Trompeta et al., 2019 ). It was described in Banti et al. (2020), Ketikidis et al. (2020) and Rossos et al. (2020). This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH–CREATE–INNOVATE (project code: T1EDK-02990) and it has been financially supported by the State Scholarships Foundation (IKϒ) (Project No. 2019-050-0503-17816), through the Operational Programme “Human Resources Development, Education and Lifelong Learning” in the context of the project “Reinforcement of Postdoctoral Researchers - 2nd Cycle” (MIS-5033021), which is co financed by Greece and the European Union (European Social Fund- ESF). C.N.B. and S.K.H. are thankful to the COST Action CA17104 "New diagnostic and therapeutic tools against multidrug resistant tumors" members for the stimulating discussions. S.K.H. acknowledges the Oncology Department of Novartis Hellas S.A.C.I. for the financial support (Project No. 82819).

Competing interests

The authors declare no conflicts of interest or competing interests.

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