WST-8 sodium

water-soluble tetrazolium dye

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].

n 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.

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]
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]
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.

[1]. Tiwari K, et al. 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.

C20H13N6NAO11S2

600.4648

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);

White to yellow or light yellow crystalline powder

267Ų

O=S(C1=CC=C(C2=NN(C3=CC=C([N+]([O-])=O)C=C3)[N+](C4=CC=C([N+]([O-])=O)C=C4OC)=N2)C([S-](=O)([O])=O)=C1)(O[Na])=O

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 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.)