Mice were infected with a luciferase-expressing strain of P. chabaudi (PccASluc [45 (link)]) as described above but kept on a reverse light cycle, as sequestration occurs during the dark cycle [45 (link)]. At the time of maximum sequestration (12.00 – 14.00 h coordinated universal time (UCT), reverse light) mice were sacrificed and perfused systemically by injection of 10 ml PBS into the heart. Organs were harvested and 0.1 g of tissue was transferred to a Precellys homogenizer tube in PBS and dissociated for one cycle, 10 seconds at 4500 rpm in a Precellys Evolution Homogenizer. The sample was then diluted 1:10 in PBS, and an equal volume (100 μL) of Bright-Glo substrate (Promega) was added. Luciferase activity was measured after 2 min incubation using a Perkin Elmer VICTOR X Light Multilabel Plate Reader.
Victor x light multilabel plate reader
Manufactured by PerkinElmer
The VICTOR X Light Multilabel Plate Reader is a versatile laboratory instrument designed for high-throughput multimode detection. It can perform absorbance, fluorescence, and luminescence measurements across a range of microplate formats.
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2 protocols using victor x light multilabel plate reader
1
Quantifying Parasite Sequestration In Mice
2
Quantification of moxGFP and Luciferase
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Quantification of mean moxGFP fluorescence was performed using flow cytometry. Luciferase expression was monitored by a luminescence-based assay. For this assay, 10 μL cell culture supernatant was plated on a white 96-well plate and combined with 100 μL PBS-T (0.1% Tween 20) containing a 1:1,000 dilution of 1 mM Coelenterazine N stock solution (PJK Biotech) dissolved in acidified methanol. Luminescence was detected directly afterward using a VICTOR X Light Multilabel plate reader (PerkinElmer).
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