Liveblazer fret b g loading kit with ccf2 am

DCs were plated into 96-well plates (1 × 10⁵ cells/well/100 μl) and infected with 250 ng p24/ml non-opsonized or opsonized R9Bal/β-lam. After 5 h incubation cells were washed and loaded for 1 h with CCF2-AM substrate solution according to the manufacturer's instructions (LiveBLAzer™ FRET-B/G Loading Kit with CCF2-AM, LifeTechnologies). Cells were washed again and developed for 16 h in CO₂-independent medium (Gibco) containing 10% FCS and 2.5 mM probenicid. Cleavage of CCF2 was analyzed by flow cytometry after fixation of DCs in 4% paraformaldehyde.

Ebola VLPs containing a beta-lactamase-fused VP40 protein (EBOV BlaVP40) and GP were produced in Dr. García-Sastre’s lab, as previously described^{46 (link)}. LiveBLAzer FRET–B/G Loading Kit with CCF2-AM and Opti-MEM reduced serum medium were purchased from Life Technologies (Carlsbad, CA, USA). The Ebola VLP assay were performed, as previously described^{47 (link)}. Briefly, HeLa cells were plated at 750 cells/well in 3 µL; 23 nL of the drug solution was transferred to the assay plate. The cells were treated with 1 µL/well VLP. The CCF2-AM beta-lactamase substrate was added and fluorescence intensities were measured using an Envision plate reader. The assay was run in triplicate. High throughput drug screening in Ebola VLP assay using HeLa cells was conducted similarly, as previously described^{7 (link)}.

Viral fusion was analyzed as described [29 (link)]. Briefly, day 5 DCs were plated into a 96-well plate in triplicates (1.5x10⁵ cells/100μl) in RPMI in presence of 10mM Hepes (Life Technologies) and 2mg/ml DEAE-Dextran (Sigma-Aldrich). Cells were exposed to the indicated concentrations of non-opsonized (HIV) or complement-opsonized (HIV-C) HIV-1 containing Blam-Vpr. After 3 hours, cells were washed 2 times in CO₂-independent medium (Life Technologies), re-suspended in CO₂-independent medium containing 10% FCS and loaded with the CCF2-AM substrate solution (LiveBLAzer FRET-B/G Loading Kit with CCF2-AM, Life Technologies). After 2h incubation at room temperature (dark) cells were washed 2 times in CO₂-independent medium, fixed in 4% paraformaldehyde for 30 min and respective wells were pooled for flow cytometric analysis.

Viral fusion was analyzed as described (57 (link)). Briefly, the assay relies on incorporation of a β-lactamase Vpr (BlaM-Vpr) protein chimera into the virion, and in target cells, upon fusion, the transfer is monitored by enzymatic cleavage of CCF2, a β-lactamase fluorescent dye substrate. The cleavage causes changes in fluorescence from green (520 nm) to blue (447 nm) that can be monitored by flow cytometry. Only cytoplasmic virions are detected (55 (link)). In our experiment, day 5 DCs were plated into a 96-well plate in triplicate (1.5 × 10⁵ cells/100 μl) in RPMI in the presence of 10 mM HEPES (Life Technologies) and 2 mg/ml DEAE-dextran (Sigma-Aldrich). Cells were exposed to the indicated concentrations of non-opsonized (HIV) or complement-opsonized (HIV-C) HIV-1 containing BlaM-Vpr. After 3 h, cells were washed twice in CO₂-independent medium (Life Technologies), resuspended in CO₂-independent medium containing 10% fetal calf serum (FCS), and loaded with the CCF2-AM substrate solution (LiveBLAzer FRET-B/G loading kit with CCF2-AM; Life Technologies). After 2 h of incubation at room temperature (dark), cells were washed twice in CO₂-independent medium and fixed in 4% paraformaldehyde for 30 min, and well contents were pooled for flow-cytometric analysis.

Measurement of virus cell fusion efficiency was performed using chimeric viruses containing a Vpr protein fused with the β-lactamase (Vpr-BLaM) (40 (link)). MT4R5 cells were treated 30 min with doxorubicin or CA3 and infected with different doses (ng of p24) of a Vpr-BLaM containing NL-4.3 or NL-4.3Δenv-VSV-G viruses for 4 h at 37°C in the presence or absence of enfuvirtide (T20) at 5 µM. Cells were washed and incubated for 2 h with CO2-independent medium supplemented with 10% FCS and CCF2 (LiveBLAzer FRET-B/G loading kit with CCF2-AM, Thermo-Fisher Scientific). Cells were washed and fixed with 2% PFA, and the fluorescence intensity of cleaved and uncleaved CCF2 was measured by flow cytometry on a BD LSRII. Data were analyzed using the FlowJo 10 Software (BD Biosciences).

β-lactamase (Bla) translocation was quantified as reported previously [43 (link), 44 (link)] using LiveBLAzer FRET-B/G Loading Kit with CCF2-AM (ThermoFisher Scientific). Plates were read in a SpectraMax M2 fluorometer (Molecular Devices) with a filter set 450/520 nm. See S1 Text for details.

Spleens were collected and splenocytes were isolated in a single cell suspension after being passed through a 100 micron filter. Splenocytes were then subjected to red blood cell lysis by treatment with red blood cell lysis buffer (Sigma # R7757) per manufacturer’s recommendation. Peritoneal cells were collected with 10 mLs of cold 1% FBS DMEM. β-lactamase activity was detected using the LiveBLAzer FRET-BG/Loading Kit with CCF2-AM (ThermoFischer Scientific # K1025) as previously described [17 (link),29 (link),32 (link)]. Cell surface antibodies used were CD19-AlexaFluor 700 (clone eBio1D3, eBioscience # 56-0193-81), CD38-APC (clone 90,eBiosciences #17-0382-81), IgD-APC-Cy7 (clone 11-26c.2a, Biolegend # 405716), and CD5-APC (clone 53–7.3, eBioscience # 17-0051-81). Fc blocking antibody 24G2 was used in staining to prevent antibody binding to cellular Fc receptors.