PaBrH, PaKiT, and NBF-L cells at a density of 2.5 × 105 cells/well (6-well plate) were infected with rABLV-GFP (MOI 1.0) for 48 h. At 24 hpi, rapamycin (RAPA; Sigma-Aldrich), small molecule enhancer of autophagy-28 (SMER; TOCRIS; Bristol, UK), or NVP BEZ235 (BEZ; Selleck Chemicals; Houston, TX, USA) were added to the wells and incubated with the ABLV-infected cell culture for 24 h. At 48 hpi, cell culture supernatants and whole cell lysates were collected. Whole cell lysates were processed as described above. Supernatants were centrifuged for 10 min at 2500 rpm to remove cell debris, then serially diluted and incubated with HEK293T cells at 5 × 104 cells/well (96-well plate) to determine rABLV-GFP titers; FITC anti-Rabies monoclonal globulin (Fujirebio Diagnostics) was used to quantify wt-ABLV titers after BEZ treatments. We attempted to monitor autophagy levels by flow cytometry after RAPA and SMER induction using a CYTO-ID® Autophagy detection kit (Enzo Life Sciences, Inc.; Famingdale, NY, USA), which stains autophagosomes with a green fluorescent dye; a positive stain was confirmed using the PaKiT cell line.
Fitc anti rabies monoclonal globulin
Manufactured by Fujirebio
Sourced in United States, Japan
The FITC anti-rabies monoclonal globulin is a laboratory reagent used for the detection and identification of rabies virus. It is a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody that specifically binds to the rabies virus antigen, allowing for its visualization under a fluorescence microscope.
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Lab products found in correlation
Nvp bez235, by Selleck Chemicals (1 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (1 mentions)
Prism 9, by GraphPad (1 mentions)
96 well plate, by Corning (1 mentions)
Mab 5b12, by MyBioSource (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
10 protocols using fitc anti rabies monoclonal globulin
1
Modulating ABLV Infection via Autophagy
2
Quantifying Rabies Virus Infection Kinetics
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NA cell monolayers were infected with rHep-Flury and variant viruses at a MOI of 0.005, then incubated for a 72h period at 34°C, and stained every 12 h with FITC Anti-Rabies Monoclonal Globulin (Fujirebio, Malvern, PA, USA), before they were examined under a fluorescence microscope(Wirblich and Schnell, 2011 (link)).
3
Antiviral Assay for Rabies Virus
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In a previously developed antiviral assay [25 (link)], the fluorescent viral signal was quantified with a standard plate reader in 96-well plates. Similarly, in this study, hit compounds were serially diluted 2-fold, starting from 50 µM to yield 8 concentrations. A total of 15,000 BHK-21 cells were incubated with compounds overnight at 37 °C and 5% CO2 in a 150 µL assay medium. After the incubation, mCherry-SAD-B19 or CVS-11 strains were added at an MOI of 0.01 TCID50/cell in a volume of 50 µL and further incubated at 37 °C for 5 days. For the CVS-11 strain infection, the cells were fixed with 80% cold acetone for 30 min at 5 dpi. Following a 3-time PBS washing, RABV N protein was stained using a 1:100 diluted FITC Anti-Rabies Monoclonal Globulin (Fujirebio, Gent, Belgium). The fluorescence intensity from antibody staining (CVS-11) or mCherry expression by the virus (mCherry-SAD-B19) was quantified using a microplate reader (SPARK, Tecan, Mechelen, Belgium). Cell viability was determined by an MTS assay as described previously [26 (link)]. The program GraphPad Prism 9.0 was used to calculate the half maximal effective concentration (EC50), the 50% cytotoxic concentration (CC50), and the selectivity index (SI = CC50/EC50).
4
Virus Titration and Growth Kinetics
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Virus titre was determined in triplicate in BHK cells as previously described [32 (link)]. Cells were seeded at 3 × 105 cells per well and infected with virus serially diluted 10-fold in a 96-well plate (Corning). Cells were fixed with 80% acetone and stained with FITC anti-rabies monoclonal globulin (Fujirebio, Tokyo, Japan) at 48 h post-infection (hpi). Fluorescent foci were counted and recorded as fluorescent focus forming units per mL (ffu/mL).
Virus growth for cSN-KBLV was assessed using a multistep growth curve in comparison with the parent virus cSN as described previously [30 (link)]. Cells were then infected with each virus at a multiplicity of infection (moi) of 0.1 and at set time points (0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h) 150 µL of media was removed and frozen at −80 °C until required. After the course of the experiment, each of the time point aliquots were titrated in triplicate on fresh BHK cells as three independent titration experiments. Titres were compared using the Mann–Whitney test.
Virus growth for cSN-KBLV was assessed using a multistep growth curve in comparison with the parent virus cSN as described previously [30 (link)]. Cells were then infected with each virus at a multiplicity of infection (moi) of 0.1 and at set time points (0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h) 150 µL of media was removed and frozen at −80 °C until required. After the course of the experiment, each of the time point aliquots were titrated in triplicate on fresh BHK cells as three independent titration experiments. Titres were compared using the Mann–Whitney test.
5
Fluorescent Antibody Test for Rabies Virus
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A fluorescent antibody test (FAT) was undertaken as described previously [48 ]. Briefly, brain tissue was excised from the cerebellum and two impression smears were made on a microscope slide. Slides were left to air dry for 5 minutes at room temperature before being fixed in 99–100% acetone, washed, rinsed, and stained using FITC anti-rabies monoclonal globulin (Fujirebio, 800-092). Fluorescent green foci would indicate the presence of the N protein thereby indicating virus infection. Positive control material consisted of previously extracted CVS-infected mouse brain. Two mice from each group were tested.
Histopathology and immunohistochemistry were performed as described previously [49 (link)]. Mouse carcasses were fixed in 10% neutral buffered formalin for 2 weeks before being sectioned coronally and processed into paraffin tissue blocks. Serial sections of 4 µm were prepared and stained with haematoxylin and eosin (H&E) for histopathology or immunohistochemistry for rabies virus nucleoprotein using a monoclonal antibody, mAb 5B12 (MyBioSource Inc., San Diego, CA, USA).
Histopathology and immunohistochemistry were performed as described previously [49 (link)]. Mouse carcasses were fixed in 10% neutral buffered formalin for 2 weeks before being sectioned coronally and processed into paraffin tissue blocks. Serial sections of 4 µm were prepared and stained with haematoxylin and eosin (H&E) for histopathology or immunohistochemistry for rabies virus nucleoprotein using a monoclonal antibody, mAb 5B12 (MyBioSource Inc., San Diego, CA, USA).
6
Viral Titre and Growth Kinetics Determination
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The titre of cultured viruses was determined in focus-forming units per mL (FFU/mL) as described previously [42 (link)]. Briefly, BHK-21 cells were seeded and 10-fold serially diluted RABV was added in triplicate. After fixation with 80% acetone and staining with FITC anti-rabies monoclonal globulin (Fujirebio), an FFU/mL value was calculated.
To investigate virus growth kinetics, multistep growth curves were performed as described previously [43 (link)]. Briefly, BHK-21 cells were cultured in 12-well plates and RABV added at a moi of 0.01. After one hour, the cell monolayer was washed and media replaced. Media samples were taken at 0, 6, 12, 24, 48, 72, 96, and 120 h and immediately stored at −80 °C. After all samples had been taken and stored at −80 °C, virus titration was undertaken.
To investigate virus growth kinetics, multistep growth curves were performed as described previously [43 (link)]. Briefly, BHK-21 cells were cultured in 12-well plates and RABV added at a moi of 0.01. After one hour, the cell monolayer was washed and media replaced. Media samples were taken at 0, 6, 12, 24, 48, 72, 96, and 120 h and immediately stored at −80 °C. After all samples had been taken and stored at −80 °C, virus titration was undertaken.
7
Viral Growth and Spread Kinetics
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To investigate the virus growth, confluent NA cell monolayers were infected with one of the rescued viruses at a MOI of 0.01 for multi-step growth curves or 3 for single-step growth curves. The infected cells were maintained at 34 °C with fresh RPMI 1640 containing 5% FBS, and the culture medium were harvested at the indicated time points, the virus was titrated in triplicate on NA cells via direct fluorescent antibody assays as described previously [24 (link)]. For the viral spread, the NA cells were infected with rescued viruses at a MOI of 0.005 for 72 h incubation in total at 34 °C, then the infected cells were observed under a fluorescent microscope with the FITC anti-rabies monoclonal globulin (Fujirebio, Malvern, PA, USA) every 12 h [25 (link)].
8
Quantification of Rabies Virus Antigen
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MNA cells were seeded (4.0 × 104 cells/well) in 96-well plates and incubated overnight at 37 °C with 5% CO2. Subsequently, serial 10-fold dilutions of the virus or the 10% brain emulsion in phosphate-buffered saline (PBS) were inoculated into the cells, which were then incubated at 37 °C for 2 days. The cells were fixed with 80% acetone for 30 min and stained with fluorescein isothiocyanate (FITC) anti-rabies monoclonal globulin (FUJIREBIO, Tokyo, Japan). Antigen-positive foci were counted under a fluorescence microscope (Nikon, Tokyo, Japan) and quantified in focus-forming units (FFU) per milliliter.
9
Propagation and Characterization of Rabies Virus Strains
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Viruses (CVS11 strain, SRV9‐eGFP strain) were preserved in the authors’ lab. The virus was propagated, titered by N2a cells and stored in a −80 °C refrigerator. N2a (mouse neuroblastoma) and BSR (cloned from BHK‐21) cells were cultured in DMEM (GIBCO, USA) supplemented with 10% fetal bovine serum (GIBCO, USA), 100 units/mL penicillin and 100 µg mL−1 streptomycin sulfate. FITC anti‐rabies monoclonal globulin (Fujirebio, USA). IL‐6 monoclonal antibody (Proteintech, USA). All animals were used according to the protocol of the Scientific Ethics Committee of Chinese laboratory animals. The agreement was approved by the Animal Welfare and Ethics Committee of the Institute of Veterinary Medicine of the Changchun Veterinary Research Institute (Laboratory Animal Care and Use Committee Authorization, permit number IACUC of AMMS‐11‐2022‐041).
10
Viral Propagation and Titration Assays
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LASSARAB, LASSARAB-ΔG, FILORAB1, rVSV-GPC, and SPBN viruses were grown and titered on Vero cells. For virus production, Vero cells were cultured with Opti-Pro serum-free media supplemented with 1% P/S and 4 mM L-Glutamine (Gibco®) and inoculated with a multiplicity of infection (MOI) of 0.01 of each respective virus. Viruses were harvested up to a total of six times with media replacement (Opti-Pro) or until 80% cytopathic effect was detected. Tittering was performed by limiting dilution focus-forming assay using RABV N with 1:200 dilution of FITC anti-rabies monoclonal globulin (Fujirebio®; catalogue number: 800-092). rVSV-GPC titers were determined by plaque forming assay using 2% methyl cellulose overlay.
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