Serial dilutions of ADCs, antibodies, or small molecules were added to 0.3 × 106 cells in 0.1 mL of media and incubated for 16 h. In some cases, 25 ng/mL PFO was used to permeabilize cells and facilitate the entry of small molecules. 20 μL of media was mixed with 100 μL of 1 μM native coelenterazine (Gold Biotechnology, St Louis MO) in PBS as substrate and luminescence was measured immediately.
Native coelenterazine
Manufactured by GoldBio
Native coelenterazine is a naturally occurring luciferase substrate used in bioluminescence applications. It serves as a core component for generating light in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using native coelenterazine
1
Cytotoxicity Assay for ADCs
2
HeLa Cell Lysate Calcium Measurement
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For cell lysate
preparation, HeLa-cyt-AEQ cells were grown in 100 mm culture dishes
(Falcon, Cat. 353003). At confluence, cells were washed twice in PBS
and scraped in 250 μL of a buffer containing (in mM): 150 Tris,
0.8 phenylmethylsulfonyl fluoride (PMSF), and 0.1 ethylenediaminetetraacetic
acid (EDTA), pH 7.2. After three cycles of freeze-thawing, cells were
centrifuged (12,000g, 5 min at 4 °C), the pellet
was discarded while the supernatant was aliquoted and stored at −80
°C.
For AEQ reconstitution, 100 μL aliquots of HeLa-cyt-AEQ
lysate were supplemented with 140 mM β-mercaptoethanol (Sigma,
Cat. M6250) and 5 μM native coelenterazine (GoldBio, St. Luis,
MO, Cat. CZ5) and allowed to reconstitute O.N. (15–24 h) on
ice. For Ca2+ measurements, serial dilutions were prepared
in 150 mM Tris, supplemented with 10 mM EDTA, pH 7.2.
For calcium
measurements, 500 μL of cell lysate was aliquoted
into the acquisition chamber. The latter was mounted on the top of
SiPM using optical grease to assure optimal matching of the refractive
index. After 30 s of acquisition, 1 mL of 150 mM Tris supplemented
with 50 mM CaCl2 was injected in the acquisition chamber.
After AEQ discharge, the trace was recorded until return to baseline
(about 4 min).
preparation, HeLa-cyt-AEQ cells were grown in 100 mm culture dishes
(Falcon, Cat. 353003). At confluence, cells were washed twice in PBS
and scraped in 250 μL of a buffer containing (in mM): 150 Tris,
0.8 phenylmethylsulfonyl fluoride (PMSF), and 0.1 ethylenediaminetetraacetic
acid (EDTA), pH 7.2. After three cycles of freeze-thawing, cells were
centrifuged (12,000g, 5 min at 4 °C), the pellet
was discarded while the supernatant was aliquoted and stored at −80
°C.
For AEQ reconstitution, 100 μL aliquots of HeLa-cyt-AEQ
lysate were supplemented with 140 mM β-mercaptoethanol (Sigma,
Cat. M6250) and 5 μM native coelenterazine (GoldBio, St. Luis,
MO, Cat. CZ5) and allowed to reconstitute O.N. (15–24 h) on
ice. For Ca2+ measurements, serial dilutions were prepared
in 150 mM Tris, supplemented with 10 mM EDTA, pH 7.2.
For calcium
measurements, 500 μL of cell lysate was aliquoted
into the acquisition chamber. The latter was mounted on the top of
SiPM using optical grease to assure optimal matching of the refractive
index. After 30 s of acquisition, 1 mL of 150 mM Tris supplemented
with 50 mM CaCl2 was injected in the acquisition chamber.
After AEQ discharge, the trace was recorded until return to baseline
(about 4 min).
3
Hoechst 33342 Staining and Gaussia Luciferase Measurement
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Cells were stained
in a 1:10,000 diluted Hoescht 33342 (Thermo Fisher) in cell media
for 20 min, 37 °C with 5% CO2. Images were obtained
using an EVOS Cell Imaging System (Thermo Fisher). For Gaussia luciferase
measurements, native coelenterazine (GoldBio) was dissolved in acidified
methanol (1 mg/mL) and was further diluted 1:100 in GLuc buffer: 0.1%
disodium phosphate, 5% glycerol, 150 mM sodium bromide, 1 mM EDTA,
25 mM Tris–HCL pH 8, and 2 mM ascorbic acid. Luciferase activity
was measured intracellularly; to this end, media was removed, and
cells were lysed in GLuc buffer supplement with 0.1% Triton X-100
for 5 min at room temperature. The cell lysate was added to Gluc buffer
with coelenterazine at a ratio of 1:5. Measurements were done in a
96- or 384-well white plate using a Spectramax M5 plate reader (Molecular
Devices).
in a 1:10,000 diluted Hoescht 33342 (Thermo Fisher) in cell media
for 20 min, 37 °C with 5% CO2. Images were obtained
using an EVOS Cell Imaging System (Thermo Fisher). For Gaussia luciferase
measurements, native coelenterazine (GoldBio) was dissolved in acidified
methanol (1 mg/mL) and was further diluted 1:100 in GLuc buffer: 0.1%
disodium phosphate, 5% glycerol, 150 mM sodium bromide, 1 mM EDTA,
25 mM Tris–HCL pH 8, and 2 mM ascorbic acid. Luciferase activity
was measured intracellularly; to this end, media was removed, and
cells were lysed in GLuc buffer supplement with 0.1% Triton X-100
for 5 min at room temperature. The cell lysate was added to Gluc buffer
with coelenterazine at a ratio of 1:5. Measurements were done in a
96- or 384-well white plate using a Spectramax M5 plate reader (Molecular
Devices).
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