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The kits were added in December 1999.
The standard cytokines included in the ELISA kits were added to their respective plates in accordance with the manufacturer's instructions and the plates were then incubated for another 24 hours at 37°C in 5% CO2.
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250 μL of SOC medium (provided with the kit) were added to the cells and horizontally agitated (200 rpm) for 1 h at 37 °C.
For the negative and positive controls, 5 μl PCR-grade H2O and 5 μl foodproof® Alicyclobacillus Control Template respectively (provided in the kit) were added to the premixed commercial PCR reagent.
To extract DNA, one 5 mm stainless steel bead and 180 µL buffer ATL (from Qiagen's DNeasy Blood and Tissue kit) were added to each CVS sample.
After 10 minutes of incubation, 10 µL of stabilizer solution (provided in the Pi color Lock TM ALS kit) were added in each well.
5 volumes of PB buffer (supplied in kit) were added to 1 volume of the PCR product and the solution added to a QIAquick spin column in a 2 ml collection tube.
To assess lytic ATP release, the cytotoxicity detection kit (Roche Diagnostics, Germany, Mannheim) was used according to the manufacture's instructions.[29] In short, confluent HMEC-1 monolayers were exposed to normoxic or hypoxic (2% oxygen) conditions over indicated time periods, then 100 µl of the substrate mixture from the kit were added to 100 µl of the collected protein-free supernatant.
Positive and negative controls provided with the kit were added to two wells per plate.
Anti-DNA-POD and anti-histone solution of the Kit were added.
Reagent R1 and NaOH from the assay kit were added to the supernatant.
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