Lipofectamine™ MessengerMAX™ (Life Technologies) was diluted in opti-MEM to the desired working concentration and dispensed onto 384-well white assay plates (Greiner 781080). A source plate (Labcyte LP-0200) containing serial dilutions of the mRNAs was prepared using the Bravo liquid handler (Agilent), and a 10-point 2-fold dose titration of each mRNA was dispensed onto the assay plate using Echo555 (Labcyte). After a 10 min incubation, 4000 MIA PaCa-2 or SJCRH30 cells or 6000 BJ cells were added per well. For kinetic monitoring, 20 μM Endurazine (Promega), an extended time-released live cell substrate, was added to each well. Luminescence was measured continuously at 1 h intervals for 48 h on the Tecan Spark 10M set to 37ºC, 5% CO2. For end-point HiBiT protein detection, the NanoGlo HiBiT lytic detection assay (Promega, N3040) was performed as per the manufacturer's instructions. Luminescence signal was determined using the Envision plate reader, and values were normalized to a HiBiT-control protein (Promega, N3010)
Endurazine
Manufactured by Promega
Sourced in United States
Endurazine is a lab equipment product from Promega. It is designed to provide long-lasting, stable performance for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Echo 555, by Beckman Coulter (2 mentions)
Lipofectamine messengermax, by Thermo Fisher Scientific (2 mentions)
Nanoglo hibit lytic detection assay, by Promega (2 mentions)
N3010, by Promega (2 mentions)
Bravo liquid handler, by Agilent Technologies (2 mentions)
Celsens software, by Olympus (1 mentions)
Ix83 zdc, by Olympus (1 mentions)
Dynasore, by Merck Group (1 mentions)
Nystatin, by Merck Group (1 mentions)
Bcye growth supplements, by Thermo Fisher Scientific (1 mentions)
Nanoglo live cell buffer, by Promega (1 mentions)
Spark plate reader, by Tecan (1 mentions)
5 protocols using endurazine
1
Transient mRNA Transfection Assay
2
Imaging Dynamic Dll1 Expression in Muscle Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze dynamic Dll1 expression in adult and embryonic muscle stem cells in wild-type, MyoD−/− and TxHes1 genetic backgrounds, the Dll1luc allele was used. For analysis of dynamic Dll1 expression in Dll1type2 mutant cells or embryos, luciferase produced by the Dll1type2 allele was imaged. For analysis of dynamic Dll1 expression in spheres, expression of Nanoluc produced by the EpDll1-NanoLuc indicator plasmid was used.
For imaging, myofibers were incubated in 35-mm glass-bottom dishes at 37 °C in 5% CO2, and 1 mM luciferin was added to the culture medium immediately before imaging. For NanoLuc imaging, 100× diluted Endurazine (Promega, Wisconsin, USA) was added to the medium. Bioluminescence images were acquired by an inverted microscope (IX83-ZDC, Olympus, Tokyo, Japan) with a cooled EM-CCD camera (EM-X2 C9100-23B, Hamamatsu, Shizuoka, Japan) in a dark room. The filters and camera control were adjusted automatically using the CelSens software (Olympus, Tokyo, Japan). Frames were acquired with exposure times that were adjusted to the expression levels, i.e., 6–9 min exposure time for the luminescence signals25 (link),62 .
For imaging, myofibers were incubated in 35-mm glass-bottom dishes at 37 °C in 5% CO2, and 1 mM luciferin was added to the culture medium immediately before imaging. For NanoLuc imaging, 100× diluted Endurazine (Promega, Wisconsin, USA) was added to the medium. Bioluminescence images were acquired by an inverted microscope (IX83-ZDC, Olympus, Tokyo, Japan) with a cooled EM-CCD camera (EM-X2 C9100-23B, Hamamatsu, Shizuoka, Japan) in a dark room. The filters and camera control were adjusted automatically using the CelSens software (Olympus, Tokyo, Japan). Frames were acquired with exposure times that were adjusted to the expression levels, i.e., 6–9 min exposure time for the luminescence signals25 (link),62 .
3
Transient mRNA Transfection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipofectamine™ MessengerMAX™ (Life Technologies) was diluted in opti-MEM to the desired working concentration and dispensed onto 384-well white assay plates (Greiner 781080). A source plate (Labcyte LP-0200) containing serial dilutions of the mRNAs was prepared using the Bravo liquid handler (Agilent), and a 10-point 2-fold dose titration of each mRNA was dispensed onto the assay plate using Echo555 (Labcyte). After a 10 min incubation, 4000 MIA PaCa-2 or SJCRH30 cells or 6000 BJ cells were added per well. For kinetic monitoring, 20 μM Endurazine (Promega), an extended time-released live cell substrate, was added to each well. Luminescence was measured continuously at 1 h intervals for 48 h on the Tecan Spark 10M set to 37ºC, 5% CO2. For end-point HiBiT protein detection, the NanoGlo HiBiT lytic detection assay (Promega, N3040) was performed as per the manufacturer's instructions. Luminescence signal was determined using the Envision plate reader, and values were normalized to a HiBiT-control protein (Promega, N3010)
4
Cellular Entry Mechanisms Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The day before infection, a 96-well black-wall clear-bottom plate was seeded with 1.5 × 104 cells/well. Entry inhibitors were pre-incubated with cells for 30 min at 37 °C; then Endurazine (Promega, Madison, WI, USA) was added to the cells at the time of infection. HAP1 cells were infected at an MOI of 1 and HΔDAG1 cells were infected with 100 times more virus to reach similar infection rates. Luminescence was measured as described above. Entry inhibitors: 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) (50 µM), dynasore (6.25 µM), nystatin (30 µg/mL), chlorpromazine hydrochloride (0.625 µg/mL) (all from Millipore Sigma, Burlington, MA, USA), dissolved in either dimethyl sulfoxide (DMSO) or water.
5
Legionella pneumophila Effector Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HiBiT-tagged effector proteins were overexpressed in L. pneumophila using the plasmid pxDC61. One day prior to infection, RAW 264.7 macrophages expressing LgBiT [28 (link),29 (link)] were seeded in a white clear-bottom 96 well cell culture plate (Greiner) at 8 x 104 cells/well. L. pneumophila strains were grown for 2-3 days on BCYE-Agar plates supplemented with “BCYE Growth Supplements” (Oxoid) and appropriate antibiotics. Gene expression was induced by incubation of bacteria at 37 °C on BCYE agar plates with 0.5 mM IPTG for 24 hours. 100 μl of bacteria resuspended in HBSS + DrkBiT (1:1000) at 1.6x108 cfu/ml were used to infect the RAW 264.7 macrophages (MOI = 200). After centrifugation at 300 x g for 8 minutes, 25 μl of NanoGlo® live cell buffer (Promega) supplemented with 1:20 of the extended live cell substrate Endurazine™ (Promega) was added to each well. Luminescence was measured at 37°C and 5 % CO2 every 15 minutes for 12 hours in a Tecan Spark plate reader.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!