Team:Debrecen-Hungary/protocols/Ligand Treatment

Scientific Background

Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay.

Overview

The target cells in our case are COS1 cells. Performing the protocol from the beginning to the end takes no more than 1 hour, and the following steps are included:

1. Preparation of the ligand solutions

2. Removal of the transfection medium

The goal is to get rid of the transfection medium from the COS1 cells 5-7 hours after transfection, because:

- PEI can damage the cells

- FBS Free medium used for transfection can cause starvation

3. Cell refeeding with Ligand-containing medium

Our aim is to refeed the cells with 10% FBS containing DMEM and also give appropriate amounts of Ligand in one step.
The 10% FBS in the medium will provide the necessary proteins and other molecules for proliferation and for life
functions. 
The ligand will activate the nuclear receptor and will promote the gene expression.

Preparation of the ligand solutions

Materials

- Nuclear receptor-transfected cells in a 48-well plate

- 10% FBS containing DMEM medium

- Ligand solution in known concentration

- 50 ml, 15 ml tubes

- Pasteur pipettes, pipettor, serological pipettes, pipettes, pipet tips

- Sterile laminar air flow box with flame and vacuum system, 37°C waterbath, 37°C incubator, 70% ethanol squirt bottles

Procedure

- in the case of 1-type receptor transfection (GAL4PPARg):

We use different concentrations of ligand, so as to make a dose-response curve in the subsequent measuring process:

10 μM: 200 * (x/10) μL Ligand up to 200μL with 10% DMEM /per well/

2 μM: 200 * (x/2) μL Ligand up to 200μL with 10%FBS DMEM /per well/

0,4 μM: 200 * (x/0,4) μL Ligand up to 200μL with 10%FBS DMEM /per well/

0,08 μM: 200 * (x/0,08) μL Ligand up to 200μL with 10%FBS DMEM /per well/

0,016 μM: 200 * (x/0,016) μL Ligand up to 200μL with 10%FBS DMEM /per well/

0 μM(negative control): 1 μL DMSO-ethanol(1:1) up to 200μL with 10%FBS DMEM /per well/

if

x = original ligand concentartion[mM]

we use 48 well plates

DMSO-ethanol is the dissolvent of the ligand

-Prepare the Ligand dilutions:

- We use 6 wells for 1 concentration / plate

- the total concentration of one well is 200 μL

- For this reason, from 1 concentration we need 6x200=1200 μL

We are doing serial dilutions (5-fold-dilutions) in 15 ml tubes:

1st dilution (10μM): I calculate with 5000 ul, put 5000*(x/10) μL Ligand solution and fill it up to 5000 μL with 10% FBS containing DMEM. Mix it with pipetting.

2nd dilution (2μM): 1 ml from the 1st dilution + 4 ml DMEM 10%

3rd dilution (0,4μM): 1 ml from the 2nd dilution + 4 ml DMEM 10%

4th dilution (0,08μM): 1 ml from the 3rd dilution + 4 ml DMEM 10%

5th dilution (0,016μM): 1 ml from the 4th dilution + 4 ml DMEM 10%

6th solution (0 μM): 6 μL-ethanol 1:1 solution + 4,994 ml DMEM 10%

• you can use serological pipettes and pipettor, or micropipettes with sterile pipette tips.

• these solutions will be enough for three 48-well plates, if you have less than 3 plates you can store the tubes at +4°C and use them next time.

- in the case of a 2-type receptor transfection:

The receptor to be activated is Vitamin D Receptor (VDR) and the Ligand is Vitamin D. We use 10e-8 μM Vitamin D. We treat 24 wells only, the other 24 will be negative controls.

-Prepare the Vitamin D solution in a 15 ml tube:

Fill 2000 / (original cc. of the ligand solution [mM]/10e-8) μL ligand solution up to 2000 μL with 10% FBS containing DMEM. Use micropipettes and sterile pipet tips.

Removal of the transfection medium

Materials

- Nuclear receptor-transfected cells in a 48-well plate

- Pasteur pipettes

- Sterile box with flame and vacuum system

Procedure

Steps:

1. Prepare the hood : put on your gloves, turn on the ventillation (it needs 15 minutes to filter the air in the box), clean the inner space of the hood with 70% ethanol. Spray hands with ethanol.

2. Place the plate into the hood.Turn on the vacuum system, insert the Pasteur pipette into the vacuum tube. Fire-sterilize the Pasteur pipette.

3. Aspire the used medium from all of the wells, pay attention so as not to aspire the cells (touch only the bottom-side of the wells).

4. Put back the cap of the plate, we don’t want the cells to go dry.

Cell refeeding with Ligand-containing medium

Materials

- Ligand solutions prepared in point 1.

- 48 well plate without medium (point 2.)

- 10% FBS containing medium

- Repeating pipet and tips

- 37°C incubator, 37°C waterbath, Sterile laminar air flow box, 70% ethanol squirt bottles

Procedure

Steps:

1. Warm up the Ligand solutions for 1- receptor transfected cells and the Ligand solution + 10% FBS DMEM for 2-receptor transfected cells

2. You use the same hood which you prapared: put everything what is needed into the hood after spraying down with 70% alcohol.

3. Take down the cap of the plate. Vortex the Ligand solutions, after that put 200-200 ul from these solutions into the adequate wells quickly (in other case the cells will go dry). Release the solutions onto the wall of the wells

After 1-receptor transfection - by using a 200 μl or a 1 ml pipet with sterile tips

10 mM: A1-A3 and E1-E3

2 mM: A4-A6 and E4-E6

0,4 mM: B1-B3 and F1-F3

0,08 mM: B4-B6 and F4-F6

0,016 mM: C1-C3 and G1-G3

0 mM: C4-C6 and G4-G6

After 2-receptor transfection – by using a repeating pipet

• Ligand treated /well A1-D6/ : 200 ul 10e-8 mM Ligand- DMEM solution

• Non-treated /well E1-H6/ : 200 ul "0 μM" solution

4. Put the „treated” COS1 cells into the 37°C incubator for 2 days, after 2 days comes the Luciferase assay to make sure that the transfection worked and to measure the Nuclear Receptor’s response to the ligand.

5. Clean the surface

Notes & troubleshooting

References

1. Junn Yanagisawa, Yasuo Yanagi, Yoshikazu Masuhiro, Miyuki Suzawa, Michiko Watanabe, Kouji Kashiwagi, Takeshi Toriyabe, Masahiro Kawabata, Kohei Miyazono, Shigeaki Kato "Convergence of Transforming Growth Factor- and Vitamin D Signaling Pathways on SMAD Transcriptional Coactivators" [http://www.sciencemag.org/cgi/content/abstract/283/5406/1317 Science 26 February 1999]

Video