GENSCRIPT TM0449 Technical Manual
Human Recombinant Melatonin MT2 Receptor Stable Cell Line
Technical Manual No. TM0449 Version 06042010
I
Introduction ….…………………………………………………………………………….
1
II
Background………..……………………………………………………………………….
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III
Representative Data………………………………………………………………………
2
IV
Thawing and Subculturing………………………………………………………………
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V
References ……………………………………………………………………………….
3 Limited Use License Agreement…………………………………………………………
4
I. Introduction
Catalog Number: M00312
Cell Line Name: CHO-K1/MT2/Gα15
Gene Synonyms: MTNR1B
Expressed Gene: Genbank Accession Number NM_005959; no expressed tags
Host Cell: CHO-K1/Gα15
Quantity: 2 vial (3×106 per vial) frozen cells
Stability: 16 passages
Application: Functional assay for MT2 receptor
Freeze Medium: 45% culture medium, 45% FBS, 10% DMSO
Complete Culture Medium: Ham’s F12, 10% FBS
Culture Medium: Ham’s F12, 10% FBS, 400 μg/ml G418, 100 μg/ml Hygromycin B
Mycoplasma Status: Negative
Storage: Liquid nitrogen immediately upon delivery
II. Background
Melatonin is a neurohormone that plays a key role in the synchronisation of circadian and seasonal functions with
cyclic environmental variations. In mammals, two melatonin receptors, MT1 and MT2, have been cloned.
Activation of MT2 melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic
nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds,
and enhance immune responses.
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III. Representative Data
-13 -12 -11 -10 -9 -8 -7 -6 -5
0
10
20
30
40
50
CHO-K1/MT2/G15
CHO-K1
EC50= 7.31 nM
Log[Melatonin] M
RFU
Figure Intracellular calcium response from CHO-K1 cells stably expressing human MT2 receptor and from
untransfected control cells. Cells were loaded with Calcium-4 then stimulated with the indicated concentrations of
Melatonin. Calcium responses were recorded on a FlexStation plate reader. Data represent the average +/standard deviation of triplicate determinations.
IV. Thawing and Subculturing
Thawing: Protocol
1. Remove the vial from liquid nitrogen tank and thaw cells quickly in a 37°C water-bath.
2. Just before the cells are completely thawed, decontaminate the outside of the vial with 70% ethanol and
transfer the cells to a 15 ml centrifuge tube containing 9 ml of complete growth medium.
3. Pellet cells by centrifugation at 200 x g force for 5 min, and discard the medium.
4. Resuspend the cells in complete growth medium.
5. Add 2 ml of the cell suspension per well in a 6 well-plate.
6. Add Hygromycin B and G418 to concentrations of 100 μg/ml and 400 μg/ml respectively the following day.
Subculturing: Protocol
1. Remove and discard culture medium.
2. Wash cells with PBS (pH=7.4) to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 ml of 0.05% (w/v) Trypsin- EDTA (GIBCO, Cat No. 25300) solution to 10 cm dish and observe the
cells under an inverted microscope until cell layer is dispersed (usually within 3 to 5 minutes).
Note: To avoid clumping, do not agitate the cells by hitting or shaking the dish while waiting for the cells to
detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting, centrifuge the cells 200 x
g force for 5min, and discard the medium.
5. Resuspend the cells in complete growth medium with Hygromycin B and G418 and add appropriate aliquots of
the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.
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