Spectra max m2 luminometer

Binding of annexin V to translocated phospholipid phosphotidylserine (PS) allows for the detection and analysis of apoptotic cells [57 (link)–59 (link)]. These assays were performed using the Enhanced Apoptosis Kit as indicated by the manufacturer (Cayman Chemical Co.) with a few modifications. Briefly, C6/36 clonal cell lines stably expressing the CHIKV-, DENV-, CHIKV/DENV, and DENV/CHIKV dual targeting intron-ΔN Bax bearing constructs were infected with DENV serotypes 1 through 4 (MOI = 0.1) or CHIKV strain 181/25 (MOI = 0.01) and assayed for annexin V binding at 24 hpi (with CHIKV) or 48 hpi (with DENV). 1x10⁶ clonal cells were scraped and placed in a well of a 96 well black opaque microtiter plate in triplicate for each clonal cell type assayed. FITC-annexin V stained microtiter plates were assayed for FITC-annexin V binding at 485 nm with the Spectra max M2 luminometer (Molecular Devices) and analyzed with Softmax Pro 5.4.5.
Three wells per clonal cell population indicated were infected with the arbovirus shown and analyzed for annexin V-FITC staining. This experiment was performed three times to give a total of nine replicates for each dual -targeting intron-ΔN Bax tested. Assay were performed in triplicate. Error bars indicate standard deviation of three independent experiments.

Binding of annexin V to translocate the phospholipid phosphotidylserine (PS) allows for the detection and analysis of apoptotic cells
[51 (link),56 (link),57 (link)]. These assays were performed using the Annexin V FITC Assay Kit as indicated by the manufacturer (Cayman Chemical Co.) with a few modifications. Briefly, C6/36 clonal cell lines stably expressing αDENV-U143-FL, αDENV-ΔU143-ΔN Bax or αDENV-U143-ΔN Bax ΔN Bax and wild type C6/36 cells were infected with each DENV serotype (MOI = 0.1). At 48 hpi 1 × 10⁶ clonal cells were scraped and placed in a well of a 96 well black opaque microtiter plate in triplicate for each clonal cell type assayed. FITC-annexin V microtiter plates were assayed for FITC-annexin V binding at 485 nm with the Spectra max M2 luminometer (Molecular Devices) and analyzed with Softmax Pro 5.4.5. Uninfected clonal and wild type C6/36 cells were also assayed as an additional negative control. Assays were performed in triplicate. Error bars indicate standard deviation of three independent experiments.

Further validation of apoptosis induction was performed by assaying for increases in effector caspase and other DEVD-specific protease activities using the EnzChekCaspase-3 Assay Kit #2 Kit (Life Technologies) as directed by the manufacturer. Briefly, C6/36 clonal cell lines stably expressing the CHIKV-, DENV-, CHIKV/DENV, and DENV/CHIKV dual targeting intron-ΔN Bax bearing constructs were infected with DENV serotypes 1–4 (MOI = 0.1) or CHIKV strain 181/25 (MOI = 0.01) and assayed for effector caspase activity at 4d.pi (for DENV) or 3dpi (for CHIKV). 1x10⁶ clonal cells were lysed, cell debris was pelleted, and lysates were placed in a well of a 96 well black opaque microtiter plate in triplicate for each clonal cell type assayed. Following addition of the Z-DEVD–R110 substrate, microtiter plates were assayed for effector caspase activity at 496 nm with the Spectra max M2 luminometer (Molecular Devices) and analyzed with Softmax Pro 5.4.5.
Three wells per clonal cell population indicated were infected with the arbovirus shown and analysed for “effector caspase” activity. This experiment was performed three times to give a total of nine replicates for each dual-targeting intron-ΔN Bax tested.