water-soluble tetrazolium dye
WST-8 sodium does not have a specific biological target; it is a chemical reagent used as a colorimetric indicator of cell metabolic activity. In the presence of an electron mediator (such as 1-methoxy PMS or phenazine methosulfate), WST-8 is reduced by dehydrogenases in metabolically active cells to a water-soluble formazan dye. The amount of formazan produced is directly proportional to the number of living cells and is quantified by absorbance at 450-460 nm.

Emulsion polymerization was carried out at 48 and 72 hours to mediate the efficacy of G-CSF. A widely used MTS-based test was compared with a bioassay of the soluble tetrazolium dye WST-8 utilizing NFS-60 cells at a concentration of 7 × 105 cells/mL vs 800 IU/mL of pelletized G-CSF at 24. Furthermore, the effectiveness of several commercially available polymer-mediated G-CSF formulations was evaluated using an optimized WST-8-based dye assay. Results indicated that while evaluating the efficacy of polymerized G-CSF using the NFS-60 cell line, the WST-8-based test offered more consistency than the conventional MTS-based assay [1].
WST-8 sodium is not used as a therapeutic agent but as a research reagent for cell viability assays. It is a tetrazolium reagent that serves as a chromogenic indicator for NADH. WST-8 sodium is used in cell proliferation assays to measure the metabolic activity of cells, which correlates with cell number and viability. The compound's activity is characterized by its reduction to a water-soluble formazan dye by cellular dehydrogenases.

WST-8 sodium is not used as a therapeutic agent and does not have in vivo activity in the pharmacological sense. It is a research reagent used in ex vivo assays to assess cell viability, proliferation, and cytotoxicity in cultured cells. The compound is not administered to animals for pharmacological studies.

In vitro bioassay using NFS-60 cells[1]
The in vitro bioassay for testing the potency of PEGylated G-CSF using MTS was based on the method outlined in European Pharmacopia (European Pharmacopoeia Citation6.3). The CCK-8 assay was performed as suggested by the manufacturer’s kit manual. The NFS-60 cells were grown in RPMI-1640 media containing 10% fetal bovine serum (FBS) and 50 µg/ml gentamycin. Before seeding, the cells were washed three times with RPMI-1640, and further separated from the solution by centrifugation at 1500 rpm for 7 min. The cells were counted using the Trypan blue dye exclusion method (Colotta et al., Citation1992; Strober, Citation2001). Various NFS-60 cell concentrations ranging from 4 × 105 to 12 × 105 cells/ml were prepared in RPMI-1640 and a range of dilutions of PEGylated G-CSF were added to the cells in order to obtain the desired final concentrations ranging from 400 to 1200 IU/ml. Appropriate cell concentrations (7 × 105 cells/ml) were dispensed in 96-well tissue culture plates with 50 µl/well volumes. To the cells, appropriate dilutions of PEGylated G-CSF (i.e., 800 IU/ml, as suggested by the European Pharmacopeia) were added to the wells. Six replicates were assigned for each cell concentration with respect to each dilution or concentration of PEGylated G-CSF. Six wells of negative controls with 50 µl of RPMI-1640 instead of PEGylated G-CSF were also included in the study. An internal standard was used in this study and the potency of the internal standard was considered to be 100%.
The assay plates were incubated at 37 °C in a 5–7% CO2 incubator for 24, 48, 72, and 96 h time points. At each time interval, three plates were removed from the incubator and subjected to the MTS and WST-8 assays to study the cell proliferation. Water soluble tetrazolium dyes MTS/PMS (10 µl of 5 mg/ml) or WST-8 (10 µl as suggested in kit) were added to each well and incubated for an additional 4 h at 37 °C in a 5–7% CO2 incubator. Plates were read at absorbance (optical density) OD490 nm for MTS and OD450 nm for WST-8 in a microtitre plate reader (Molecular Devices, Silicon Valley, CA). Similarly, PEGylated G-CSF formulations from other manufacturers (Peg-Grafeel PGAS01512, Neulastim B1029B05, NeuPEG 5010043) were also tested with the same WST-8-based assay to determine the efficacy of the assay as well as the potency of the other commercially available PEGylated G-CSF preparations.
Non-cellular assays for WST-8 sodium involve its reduction by chemical reducing agents, such as NADH, in a cell-free system. The compound is incubated with varying concentrations of NADH in the presence of an electron mediator, and the production of the formazan dye is monitored spectrophotometrically at 450-460 nm. A standard curve is generated to relate absorbance to NADH concentration. This assay is used to validate the performance of WST-8 sodium as a cell viability reagent.

Study of PEGylated G-CSF concentration[1]
For the dose–response relationship of PEGylated G-CSF on NFS-60 cells, 7 × 105 cells/ml were stimulated with various concentrations of PEGylated G-CSF for 48 h and consequently proliferative responses were measured by MTS and WST-8-based assays. The concentrations of PEGylated G-CSF employed for stimulation of NFS-60 cells were 500, 600, 700, 800, 900, 1000, 1100, and 1200 IU/ml. The change in optical density (ΔOD) as a measure of proliferative responses was calculated by subtracting the OD of unstimulated cells by the OD of the stimulated cells. The dose-dependent enhancement in proliferative responses of the NFS-60 cells consequent to stimulation with PEGylated G-CSF was observed in 700–800 IU/ml in MTS assay and the narrow range of 600–700 IU/ml in WST-8 assay.[1]

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Study of cell concentration[1]
To optimize the cell concentrations for utilization in cell-based bioassay for PEGylated G-CSF, various concentrations of NFS-60 cells ranging from 4 × 105 to 12 × 105 cells/ml were seeded in 96-well cell culture plates and subsequently stimulated with 800 IU/ml of PEGylated G-CSF for 48 h. At the end of incubation period, the plates were assayed for proliferative responses by both the MTS and WST-8 assays.[1]

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Study of incubation time[1]
To measure the optimum incubation time for assessing the proliferative responses of NFS-60 cells, 7 × 105 NFS-60 cells/ml was stimulated with 800 IU/ml PEGylated G-CSF and assayed at various time intervals (24, 48, 72, and 96 h) by MTS and WST-8 assays.
In vitro cellular assays with WST-8 sodium involve culturing cells in 96-well plates and adding the WST-8 reagent to the culture medium. Cells are incubated for 1-4 hours, during which metabolically active cells reduce WST-8 to a water-soluble formazan dye. The absorbance is measured at 450-460 nm using a microplate reader. The amount of formazan produced is proportional to the number of living cells. The assay is used for cell proliferation, cytotoxicity, and drug sensitivity testing.

In vivo animal experiments are not applicable for WST-8 sodium as it is a research reagent for in vitro cell-based assays, not a therapeutic or diagnostic agent. The compound is not administered to animals for pharmacological studies. Its use is limited to cell culture and biochemical applications.

WST-8 sodium has a molecular weight of 600.47 and a molecular formula of C20H13N6NaO11S2. It is a white crystalline powder that is hygroscopic (easily absorbs moisture). Purity is typically >95-99%. The compound is soluble in water and is typically stored at room temperature in a dry place. It is stable under recommended storage conditions.

WST-8 sodium is generally considered to have low toxicity at the concentrations used in cell viability assays. Standard laboratory safety precautions should be followed when handling the compound. It may cause skin, eye, and respiratory irritation. Appropriate personal protective equipment should be used.

[1]. A sensitive WST-8-based bioassay for PEGylated granulocyte colony stimulating factor using the NFS-60 cell line. Pharm Biol. 2015 Jun;53(6):849-54.

This study found that the WST-8-based detection method was superior to the MTS-based detection method in the quantitative analysis of the proliferative response of PEGylated G-CSF to NFS-60 cells. In addition, this study also demonstrated the potential application of the WST-8-based bioassay method in other biotherapeutic proteins with human and veterinary applications. [1]

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Background: Granulocyte colony-stimulating factor (G-CSF) is commonly used to treat neutropenia caused by chemotherapy, radiotherapy and organ transplantation. In vitro experiments have confirmed that conjugating G-CSF with polyethylene glycol (PEG) can improve its efficacy. Objective: Currently, the use of tetrazolium dye and NFS-60 cell line for in vivo bioassay is recommended to detect G-CSF, but there is no such monograph in the pharmacopoeia for PEGylated G-CSF. This study evaluated the applicability of the detection method recommended for detecting granulocyte colony-stimulating factor (G-CSF) to PEGylated G-CSF. Materials and Methods: A commonly used assay based on MTS [3-(4,5-dimethylthiazolyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole] was compared with a bioassay using the water-soluble tetrazolium dye WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole]. PEGylated G-CSF at a concentration of 7 × 10⁵ cells/mL was measured at 24, 48, 72, and 72 hours to determine its efficacy. Furthermore, the efficacy of various commercially available polyethylene glycol-modified G-CSF formulations was tested using an optimized WST-8 dye-based assay. Results: The results showed that the WST-8-based detection method had higher sensitivity in determining the potency of PEGylated G-CSF using the NFS-60 cell line compared with the traditional MTS-based detection method. Conclusion: Our study suggests that the WST-8-based bioassay method has potential application value in other biotherapeutic proteins with human and veterinary applications. [1]
WST-8 sodium (CAS 193149-74-5) is a water-soluble tetrazolium salt used for assessing cell metabolic activity in cell proliferation and cytotoxicity assays. It is reduced by cellular dehydrogenases to a water-soluble formazan dye, the production of which is proportional to the number of living cells. WST-8 sodium is a widely used reagent for cell viability testing and is available from various commercial suppliers.

C20H13N6NAO11S2

600.4706

599.9981

C, 40.01; H, 2.18; N, 14.00; Na, 3.83; O, 29.31; S, 10.68

193149-74-5

193149-74-5 (sodium);755734-51-1 (free acid);

9894947

White to yellow or light yellow crystalline powder

4.052

0

13

4

40

1080

0

S(C1C([H])=C(C([H])=C([H])C=1C1=NN(C2C([H])=C([H])C(=C([H])C=2[H])[N+](=O)[O-])[N+](C2C([H])=C([H])C(=C([H])C=2OC([H])([H])[H])[N+](=O)[O-])=N1)S(=O)(=O)[O-])(=O)(=O)[O-].[Na+]

YCAKCISJXLQUEQ-UHFFFAOYSA-N

InChI=1S/C20H13N6O11S2.Na/c1-37-18-10-14(26(29)30)6-9-17(18)24-22-20(21-23(24)12-2-4-13(5-3-12)25(27)28)16-8-7-15(38(31,32)33)11-19(16)39(34,35)36/h2-11H,1H3/q-1+1

4-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl)-3-sulfobenzenesulfonate, sodium salt

WST-8; WST 8; 270078-53-0; MFCD09264687; sodium 4-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl)benzene-1,3-disulfonate; sodium;4-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate; WST-8 Sodium; WST8.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~83.33 mg/mL (~138.77 mM)
DMSO : ~10 mg/mL (~16.65 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (1.67 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (1.67 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1 mg/mL (1.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (166.54 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)