Table 1.
Protocols for High-Throughput Screen Calcium Mobilization Assay
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Plate cells | 20 mL | Stable HEK293T cell line with TET-inducible 5-HT2B receptor |
| 2 | Calcium indicator dye | 20 mL | Exchange media for 1 μM Calbryte520 AM in assay buffer |
| 3 | Incubation | 60 m | 37°C |
| 4 | Assay buffer | 20 mL | Remove dye and replace with HBSS +20 mM HEPES |
| 5 | Library compounds | 20 mL | 10 mM in DMSO stocks diluted to 2 × (20 mM) for live imaging dispense |
| 6 | Control | 20 mL | 10 mM in DMSO stock SB204741 (VU0254140) diluted fresh to 2 × (20 mM) |
| 7 | 5-HT | 10 mL | EC80 5-HT or Emax 5-HT |
| 8 | Incubation | 10 m | ambient equilibration period |
| 9 | Panoptic reader | 482ex/536em | Live kinetic imaging |
| Step | Notes | ||
|---|---|---|---|
| 1 |
|
|
Corning 384 well amine coated plates, plated with Multidrop Combi |
| 2 |
|
|
Exchange from media to assay buffer using ELX405 plate washer |
| 3 |
|
|
Lidded plates in incubator at 37°C and 5%CO2 |
| 4 |
|
|
Exchange from dye to assay buffer using ELX405 plate washer |
| 5 |
|
|
Create aliquot from DMSO stock library into aqueous 2 × working solution using ECHO OMICS |
| 6 |
|
|
Create aliquot from DMSO stock into aqueous 2 × working solution using ECHO OMICS |
| 7 |
|
|
Create aliquot from DMSO stock into aqueous 5 × working solution using ECHO OMICS |
| 8 |
|
|
Unlidded plate |
| 9 | 2 × plate with compounds, controls; 5 × plate with 5-HT; and cell plate load into Panoptic |
5-HT2B, serotonin receptor 2B; DMSO, dimethyl sulfoxide; HBSS, Hanks' Balanced Salt Solution; HEPES, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid; TET, tetracycline.