GENSCRIPT TM0420 Technical Manual

Human Recombinant Free Fatty Acid Receptor 1 Stable Cell Line
Technical Manual No. TM0420 Version 06042010
I
Introduction ….…………………………………………………………………………….
1
II
Background……………………………………………………………………………….
1
III
Representative Data………………………………………………………………………
2
IV
Thawing and Subculturing………………………………………………………………
2
V
References ……………………………………………………………………………….
3 Limited Use License Agreement…………………………………………………………
4
I. Introduction
Catalog Number: M00273 Cell Line Name: CHO-K1/FFA1/Gα15 Expressed Gene: GenBank Accession Number NM_005303; no expressed tags Host Cell: CHO-K1 Quantity: 2 vial (3×106 per vial) frozen cells Stability: 16 passages Applications: Functional assays for FFA1 receptor (GPR40) Freeze Medium: 45% culture medium, 45% FBS, and 10% DMSO
Complete Culture Medium: Ham’s F12, 10% FBS Culture Medium: Ham’s F12, 10% FBS, 200 μg/ml Zeocin, 100 μg/ml Hygromycin B
Mycoplasma Status: Negative Storage: Liquid nitrogen immediately upon delivery
II. Background
Free fatty acid G protein coupled receptor family consists of four members and plays significant roles in nutrition regulation. GPR40 (FFA1) and GPR120 are activated by medium and long-chain FFAs, whereas GPR41 and GPR43 (FFA2) can be activated by short-chain FFAs. FFA1 is preferentially expressed in pancreatic beta-cells and mediates the majority of the effects of FFAs on insulin secretion. Researches show that FFA1 is a potential therapeutic target and plays an important role in the chain of events linking obesity and type2 diabetes.
-1-
III. Representative Data
-11 -10 -9 -8 -7 -6 -5 -4 -3 -2
0
25
50
75
100
125
CHO-K1/FFA1/G15
CHO-K1
EC50= 4.4 M
Log[OA] M
RFU
Figure: Shown above are the intracellular calcium responses of CHO-K1 cells stably expressing Homo sapiens
free fatty acid receptor 1 (FFA1/GPR40) and of untransfected control cells. Cells were loaded with Calcium-4 and then stimulated with the indicated concentrations of Oleic acid. 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 Zeocin to concentrations of 100 μg/ml and 200 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 Zeocin and add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.
-2-
Loading...
+ 2 hidden pages