Assay method for the antiproliferative activity of human galectin-9

Authors：

Category

Sugar binding proteins

Protocol Name

Authors

Nishi, Nozomu *
Division of Research Instrument and Equipment, Life Science Research Center, Kagawa University

Nakamura, Takanori
Department of Endocrinology, Faculty of Medicine, Kagawa University

*To whom correspondence should be addressed.

KeyWords

Galectin T cell Lectin Apoptosis Jurkat

Reagents

●	Jurkat T lymphoma (lymphoblastoma) cells
●	RPMI 1640* supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin (RPMI 1640 medium) * With L-glutamine and sodium bicarbonate (Ex. Sigma-Aldrich R8758)
●	Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan)
●	1.2% (w/v) sodium dodecyl sulfate (SDS)

Instruments

●	CO₂ incubator
●	Clean bench
●	Tabletop centrifuge
●	Inversed phase contrast microscope
●	Microplate reader

Methods

Assay method for the antiproliferative activity of human galectin-9

1)	Collect Jurkat cells* by centrifugation at 150 × g for 5 min using the tabletop centrifuge (*see Comment).	Comment 1

2)	Resuspend the cell pellet in fresh RPMI 1640 medium.	Comment 0

3)	Determine the cell concentration with a hemocytometer.	Comment 0

4)	Add an appropriate volume of the medium to make the final concentration 3 × 10⁴ cells/90 μL.	Comment 0

5)	Inoculate the cell suspension into a 96-well tissue culture plate (90 μL/well) using a multi-channel pipette.	Comment 0

6)	Incubate the plate for 2 h at 37°C under a humidified 5% CO₂ atmosphere (CO₂ incubator).	Comment 0

7)	Add 10 μL/well of sterile test samples directly to the plate.	Comment 1

8)	Incubate the plate for 24 h* at 37°C in the CO₂ incubator (*see Comment).	Comment 1

9)	Add 10 μL/well of WST-8 reagent.	Comment 1

10)	Incubate the plate for 2 h* at 37°C in the CO₂ incubator (*see Comment).	Comment 1

11)	Add 10 μL/well of the 1.2% SDS solution.	Comment 0

12)

Determine the difference between the absorbance at 450 and that of 620 nm with the microplate reader.

Comment 0

Notes

This protocol can be used for other cell types including adherent cells. When an adherent cell line is used as a target for galectin-9, the incubation time after inoculation should be longer (up to 24 h) to facilitate the attachment of protease-treated cells.

Figure & Legends

Fig. 1. The antiproliferative effect of G9S on Jurkat cells was determined by means of the WST-8 assay.

Jurkat cells were cultured in the presence of an assay sample for 24 h. After the addition of WST-8 reagent, the culture was continued for 2 h. The viable cell number was determined with a microplate reader. The viable cell number for the untreated control culture was taken as 100%. The results represent the means ± S.D. of triplicate measurements.

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Date of registration：2014-08-26 16:37:05