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Fig. 2 | Virology Journal

Fig. 2

From: Lithium inhibits NF-κB nuclear translocation and modulate inflammation profiles in Rift valley fever virus-infected Raw 264.7 macrophages

Fig. 2

Determination of the effects of lithium on oxidative burst after Raw 264.7 cell are challenged with RVFV and expression of levels of the antioxidant enzyme. a Cells were seeded at 4 × 105 cells/well in a 6 well plate for 3 h and then inoculated with 1 × 104.8 viral titre/mL for an hour, the excess virus was substituted with fresh media and various lithium concentrations as well as 5 mg/mL LPS for 24 h. After 24 h of inoculation, cells were stained with H2DCF-DA at RT for 30 min in the dark and then cell fixed with 3.7% paraformaldehyde for an hour. The pictures where captured with EVOS FL Colour imaging system (Life Technologies, USA) Ex: 495 nm; Em: 515 nm. b Cells were seeded at 1 × 106 cells/well in a 96 well plate for 3 h and then inoculated with RVFV at 103.8 viral titter/100uL for an hour. The excess virus was then substituted with fresh media and lithium concentrations as well as 5 mg/mL LPS, this was incubated for 12 and 24 h. After the incubation hours the cells were stained with H2DCF-DA for 30 min in the dark, and then the fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Fisher Scientific, USA) at ex (485 nm)-em (538 nm). The graphs were developed with Graph Pad Prism-6 software and GraphPad InStat-3 was used to establish the statistical analysis. c In order to determine the expression of HO-1 protein, Raw 264.7 cell where seeded at 1 × 106 cell/mL for 3 h and then inoculated with 104.8 viral titre/mL for 1 h, and then the excess virus was substituted with a fresh media and lithium concentrations for 12 h. This was then followed by isolation of proteins and then western blotting assay. The pictures were captured with ChemiDoc XRS+ (Bio-RAD, USA)

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