Protocols

CELT-316 Adenosine A2A Confocal Imaging Protocol

Protocol for imaging Adenosin A2A receptors in non-fixed HCT116 colorectal tumor cells

Seed 1 ml of HCT116 cell suspension (5x104 cells/ml of McCoy + 1% Pen/Strep + 10% FBS complete medium) into the inner well of a glass bottom μ-Dish 35 (Ibidi). After cell attachment, add 1 additional ml of complete medium to ensure optimal grow conditions, and incubate them at 37°C and 5% CO2 until confluence.

Prior to cell labelling, remove the complete culture medium and wash once with FBS-free medium. Add 1ml of the Celtarys adenosine receptor fluorescent ligand CELT-316 at a final concentration of 250nM in medium without FBS. CELT-316 selectively binds to Adenosine A2A receptor with a Ki of 116.1nM.

Directly observe the HCT116 live cells on an inverted confocal microscope (Leica SP8), using a 40x oil-immersion objective (HC PL APO CS2 40x/1.30NA) (3% 552 laser beam and Hybrid (HyD3) detector). It is not necessary to wash the cells before visualization; no background was observed with the Celtarys fluorescent ligand. Staining is achieved shortly after addition of CELT-316.

Representative image of HCT116 cells stained with Celt-316. Image was taken 2min after the addition of Celt-316 to the medium.

Representative image of HCT116 cells stained with Celt-316. Image was taken 2min after the addition of Celt-316 to the medium.

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CELT-327 Adenosine A2B-A3 Confocal Imaging Protocol

Protocol for imaging Adenosin A2A-A3 receptors in non-fixed HCT116 colorectal tumor cells

Seed 1 ml of HCT116 cell suspension (5x104 cells/ml of McCoy + 1% Pen/Strep + 10% FBS complete medium) into the inner well of a glass bottom µ-Dish 35 (Ibidi). After cell attachment, add 1 additional ml of complete medium to ensure optimal grow conditions, and incubate them at 37°C and 5% CO2 until confluence.

Prior to cell labelling, remove the complete culture medium and wash once with FBS-free medium. Add 1ml of the Celtarys adenosine receptor fluorescent ligand Celt-327 at a final concentration of 100nM in medium without FBS. For visualization purposes, cells can be co-stained with a green live-cell dye (Calcein).

Directly observe the HCT116 live cells on an inverted confocal microscope (Leica SP8), using a 40x oil-immersion objective (HC PL APO CS2 40x/1.30NA) (3% 552 laser beam and Hybrid (HyD3) detector for Celt-327 and 2% 488 laser beam, PMT 492-562 detector for Calcein). It is not necessary to wash the cells before visualization; no background was observed with the Celtarys fluorescent ligands. Staining is achieved shortly after addition of Celt-327, with fluorescent levels peaking at 5-8mins.

Representative image of HCT116 cells co-stained with Celt-327 and Calcein. Images were taken 2min after the addition of Celt-327 to the medium.

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CELT-331 CB2 HCS Protocol

High Content Screening protocol for Cannabinoid Receptors assays

METHODOLOGY:

Cannabinoid CB2 receptor competition binding experiments were carried out in a CellCarrier-96 Ultra Microplate (PerkinElmer 6055302).

40.000 cells per well of a HEK-CB2 cell line were seeded 24 hours prior to assay.

The culture medium was replaced with Hank’s balanced salt solution (HBSS. Sigma H6648). In each well was incubated CELT-331 at a concentration of 30 nM.

The ligand displacement (signal inhibition) was determined in the presence of increasing concentrations of GW405833 (Sigma G1421).

Hanks balanced salt solution was used as assay buffer for all dilutions. The reaction mixture (Vt: 100 μl/well) was incubated at 37°C for 60 min. After washing (100 μl HBSS) the cells were fixed with paraformaldehyde solution 4% in PBS (Chemcruz sc-281692). A cycle of 3 washes was performed with 100 μl of HBSS and Hoechst 33342 trihydrochloride trihydrate (Invitrogen H3570) (dilution 1:2000) was added. A final wash was performed and a 100 μl/well volume of HBSS was added.

Microscopy images were obtained using an Operetta high-content analysis system (PerkinElmer).

Representative example of the competition binding between CELT-331 30 nM and GW405833.

Representative example of the competition binding between CELT-331 30 nM and GW405833.

ASSAY VALIDATION:

The HCS assay was validated using GW405833 as a reference compound binding to Cannabinoid CB2 receptor.

Fig. 1: Representation of the concentration-response curve of GW405833, antagonist for human CB2 receptor, generated from the fluorescence intensity of CELT-331, measured on Operetta high-content analysis system. The mean ± SEM (vertical bars) obtained in duplicate wells by extracting 9 images from each of them is shown.

Table 2: Value (IC50) obtained and described in the literature for the compound used to validate the assay.

Receptor Compound IC50 (nM) obtained Ki (nM) described Bibliographic Reference
hCB2 GW405833 16.6 12 Brogi et al., Eur J Med Chem 2011; 46:547-555
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Competition Binding Microscopy Protocol

Protocol for competition binding microscopy in transfected cells

Competition binding experiments were carried out in a CellCarrier-96 Ultra Microplate (PerkinElmer 6055302).

30.000 cells per well of a cell line expressing the desired receptor were seeded 24 hours prior to assay.

The culture medium was replaced with Hanks balanced salt solution (HBSS. Sigma H6648). In each well was incubated the compound tested; concentrations of the different compounds were established based on the Ki value obtained in radioligand binding assays.

The ligand displacement (fluorescent signal inhibition) was determined in the presence of a known binder at high concentration (for instance, for A3 receptor competition binding assay, MRS 1220 (Sigma M228) was used). Hanks balanced salt solution was used as assay buffer for all dilutions.

The reaction mixture (Vt: 100 μl/well) was incubated at 37°C for 60 min.

After washing (100 μl HBSS) the cells were fixed with paraformaldehyde solution 4% in PBS (Chemcruz sc-281692). A cycle of 3 washes was performed with 100 μl of HBSS and Hoechst 33342 trihydrochloride trihydrate (Invitrogen H3570) (dilution 1:4000) was added to visualize nuclei.

A final wash was performed and a 100 μl/well volume of HBSS was added.

Microscopy images were obtained using an Operetta high-content analysis system (PerkinElmer).

Representative Examples

Competition binding between Celtarys fluorescent ligand CELT-171 hA3 Adenosine receptor fluorescent antagonist and MRS 1220 in Hela-A3 cell line.

Hela cells expressing the human A3R labelled with:

A) Celtarys fluorescent ligand CELT-171 hA3 Adenosine receptor fluorescent antagonist at 2 nM (pink) and Hoechst 33342 (blue)

B) CELT-171 hA3 Adenosine receptor fluorescent antagonist at 2 nM and Hoechst 3342 (blue) preincubated with MRS1220 100 nM.

Competition binding between Celtarys fluorescent ligand CELT-331 hCB2 Cannabinoids receptor fluorescent ligand and GW405833 in HEK-CB2R cell line.

HEK cells expressing the human CB2R labelled with:

A) Celtarys fluorescent ligand CELT-331 hCB2 Cannabinoids receptor fluorescent ligand at 30 nM (pink) and Hoechst 33342 (blue)

B) CELT-331 hCB2 Cannabinoids receptor fluorescent ligand at 30 nM and Hoechst 3342 (blue) preincubated with GW405833 10 μM.

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