Alliance imager apparatus

Manufactured by Uvitec

Sourced in United Kingdom

The Alliance Imager apparatus is a laboratory equipment designed for capturing and analyzing digital images. It functions as an imaging system for various applications, providing high-quality image capture and processing capabilities.

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Lab products found in correlation

Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions) Ripa buffer, by Thermo Fisher Scientific (3 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions) β actin antibody clone ac 74, by Merck Group (3 mentions) Protease inhibitor, by Roche (1 mentions) Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions) Immobilon crescendo western hrp substrate, by Merck Group (1 mentions) Mouse anti flag antibody f1804, by Merck Group (1 mentions) Mab8929, by R&D Systems (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)

5 protocols using alliance imager apparatus

SARS-CoV-2 Spike Processing Analysis

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VeroE6 cells were seeded in 6-well plates and infected with BA.2 or BA.1 at 0.1 MOI. Cell lysates were harvested in 200 μL RIPA buffer (89901, Thermo Scientific ) at 24 h post infection for the analysis of spike processing. The samples were subjected to 8% of SDS-PAGE and transferred to the PDVF membranes, followed by blocked with 5% skim milk in PBS for 2h at room temperature and incubated with specific primary antibodies at 4°C overnight, followed by incubating with horseradish peroxidase (HRP) conjugated secondary antibodies (Thermo Fisher Scientific) for 1h at room temperature. The signal was developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (34580, Thermo Scientific, USA) and detected using Alliance Imager apparatus (Uvitec, Cambridge, UK). Full-length spike and S2 was detected with a rabbit anti-SARS-CoV-2 spike S2 antibody (40590-T62, Sino Biological) (1:5000). Nucleocapid (N) was detected with an in-house rabbit anti-SARS-CoV-2 N immune serum (1:10000) and β-actin was detected with a β-actin antibody (clone AC-74, A5316, Sigma, USA) (1:5000).

Chan J.F., Hu B., Chai Y., Shuai H., Liu H., Shi J., Liu Y., Yoon C., Zhang J., Hu J.C., Hou Y., Huang X., Yuen T.T., Zhu T., Li W., Cai J.P., Luo C., Yip C.C., Zhang A.J., Zhou J., Yuan S., Zhang B.Z., Huang J.D., To K.K., Yuen K.Y, & Chu H. (2022). Virological features and pathogenicity of SARS-CoV-2 Omicron BA.2. Cell Reports Medicine, 3(9), 100743.

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Western Blot Analysis of SARS-CoV-2 Spike Protein

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Cells in 12-well plates were lysed by 125 μl of radioimmunoprecipitation assay buffer (89901, Thermo Fisher Scientific) with protease inhibitor (4693159001, Roche, Basel, Switzerland) at 24 hours after transfection. Proteins were separated by SDS–polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride or nitrocellulose membrane. Specific primary antibodies were incubated with the blocked membranes at 4°C overnight, followed by horseradish peroxidase (HRP)–conjugated secondary antibodies (Thermo Fisher Scientific) incubation for 2 hours at room temperature. The signal was developed by the Immobilon Crescendo Western HRP Substrate (WBLUR0500, Merck Millipore, MA, USA) and detected using the Alliance Imager apparatus (Uvitec, Cambridge, UK). Mouse anti-flag antibody (F1804, Sigma-Aldrich, St. Louis, Missouri, USA) was used as the primary antibody to detect the overexpression of the flag-tagged proteases. Human ACE2 was detected with a rabbit anti-V5 antibody (CM3005, ImmunoWay, Plano, TX, USA). β-Actin was used as the loading control, which was detected by a mouse anti-human β-actin antibody (MAB8929, R&D Systems, Minneapolis, Minnesota, USA). The spike protein was detected with a rabbit anti–SARS-CoV-2 spike S2 antibody (40590-T62, Sino Biological, Beijing, China).

Chan J.F., Huang X., Hu B., Chai Y., Shi H., Zhu T., Yuen T.T., Liu Y., Liu H., Shi J., Wen L., Shuai H., Hou Y., Yoon C., Cai J.P., Zhang A.J., Zhou J., Yin F., Yuan S., Zhang B.Z., Brindley M.A., Shi Z.L., Yuen K.Y, & Chu H. (2023). Altered host protease determinants for SARS-CoV-2 Omicron. Science Advances, 9(3), eadd3867.

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Omicron Subvariants Spike Protein Analysis

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VeroE6-TMPRSS2 cells were challenged with Omicron subvariants at 0.5 MOI and lysed in RIPA buffer (89901, Thermo Fisher Scientific) at 48 hpi for Western blot analysis. The membranes were blocked with 5% milk for 2 h at room temperature and incubated with a rabbit anti-SARS-CoV-2 spike S2 antibody (40590-T62, Sino Biological, China) at 4 °C for overnight incubation, followed by detection with horseradish peroxidase (HRP) conjugated secondary antibodies (31460, Thermo Fisher Scientific) for 1 h at room temperature. Antigen signal was developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (34580, Thermo Scientific) and detected using an Alliance Imager apparatus (Uvitec, Cambridge, UK). β-actin was detected with a β-actin antibody (clone AC-74, A5316, Sigma-Aldrich) (1:5000).

Hu B., Chan J.F., Liu Y., Liu H., Chen Y.X., Shuai H., Hu Y.F., Hartnoll M., Chen L., Xia Y., Hu J.C., Yuen T.T., Yoon C., Hou Y., Huang X., Chai Y., Zhu T., Shi J., Wang Y., He Y., Cai J.P., Zhou J., Yuan S., Zhang J., Huang J.D., Yuen K.Y., To K.K., Zhang B.Z, & Chu H. (2023). Divergent trajectory of replication and intrinsic pathogenicity of SARS-CoV-2 Omicron post-BA.2/5 subvariants in the upper and lower respiratory tract. eBioMedicine, 99, 104916.

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SARS-CoV-2 Spike Cleavage Analysis

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Spike plasmids of SARS-CoV-2 D614G, Omicron BA.1, BA.2, S₁/S₂-10Del and all single mutation plasmids were transfected with Lipofectamine 3000 (L3000015, Thermo Fisher Scientific) in 293T cells. Cell lysates were harvested 24 h post-transfection for Western blot analysis. Specific primary antibodies were incubated with the blocked membranes at 4°C overnight, followed by horseradish peroxidase (HRP) conjugated secondary antibodies (62-6520, Thermo Fisher Scientific) for 1 h at room temperature. The signal was developed by SuperSignal West Pico PLUS Chemiluminescent Substrate (34580, Thermo Scientific, USA) and detected using Alliance Imager apparatus (Uvitec, Cambridge, UK). The full-length spike and S2 were detected with a rabbit anti-SARS-CoV-2 spike S2 antibody (40590-T62, Sino Biological) (1:5000). β-actin was detected with a β-actin antibody (clone AC-74, A5316, Sigma, USA) (1:5000). The cleavage ratio of the spike was quantified by ImageJ.

Hu B., Chan J.F., Liu H., Liu Y., Chai Y., Shi J., Shuai H., Hou Y., Huang X., Yuen T.T., Yoon C., Zhu T., Zhang J., Li W., Zhang A.J., Zhou J., Yuan S., Zhang B.Z., Yuen K.Y, & Chu H. (2022). Spike mutations contributing to the altered entry preference of SARS-CoV-2 omicron BA.1 and BA.2. Emerging Microbes & Infections, 11(1), 2275-2287.

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SARS-CoV-2 Spike Protein Expression and Infectivity

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293T cells were transfected with spike plasmids of SARS-CoV-2 WT (614G), BA.1, BA.2, BA.2.12.1, BA.4/5 and S₁/S₂Del. For infection experiment, VeroE6 cells were challenged with SARS-CoV-2 WT, BA.1, BA.2 or BA.5.2 at 2 MOI. Cell lysates were harvested in RIPA buffer (89901, Thermo Fisher Scientific) at 24 h post transfection or infection for Western blot analysis. The membranes were blocked with 5% milk for 2h at room temperature and incubated with a rabbit anti-SARS-CoV-2 spike S2 antibody (40590-T62, Sino Biological, China) at 4 °C for overnight incubation, followed by detection with horseradish peroxidase (HRP) conjugated secondary antibodies (31460, Thermo Fisher Scientific) for 1h at room temperature. The signal was developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (34580, Thermo Scientific) and detected using Alliance Imager apparatus (Uvitec, Cambridge, UK). β-actin was detected with a β-actin antibody (clone AC-74, A5316, Sigma–Aldrich) (1:5000).

Shuai H., Chan J.F., Hu B., Chai Y., Yoon C., Liu H., Liu Y., Shi J., Zhu T., Hu J.C., Hu Y.F., Hou Y., Huang X., Yuen T.T., Wang Y., Zhang J., Xia Y., Chen L.L., Cai J.P., Zhang A.J., Yuan S., Zhou J., Zhang B.Z., Huang J.D., Yuen K.Y., To K.K, & Chu H. (2023). The viral fitness and intrinsic pathogenicity of dominant SARS-CoV-2 Omicron sublineages BA.1, BA.2, and BA.5. eBioMedicine, 95, 104753.

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