Optimized production and fluorescent labelling of SARS-CoV-2 Virus-Like-Particles to study virus assembly and entry

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   ScreenIT

   Mar 29, 2022

   SciScore for 10.1101/2022.03.23.485575: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   To detect hACE2, cells were washed twice in PBS and incubated 1 hour at 4°C with primary goat antibody anti-human ACE2 protein (R&D AF933) (1:100).	anti-human ACE2 suggested: None
   Antibodies: Immunoblotting were performed by using the following antibodies: rabbit anti-M 1:1000 (100-401-A55, Rockland TEBU-BIO);rabbit anti-N 1:3000 (200-401-A50,	anti-M suggested: None anti-N suggested: None
   anti-GAPDH antibody coupled to HRP 1:25000 (G9295, Sigma).	anti-GAPDH suggested: (Sigma-Aldrich Cat# G9295, …

   SciScore for 10.1101/2022.03.23.485575: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   To detect hACE2, cells were washed twice in PBS and incubated 1 hour at 4°C with primary goat antibody anti-human ACE2 protein (R&D AF933) (1:100).	anti-human ACE2 suggested: None
   Antibodies: Immunoblotting were performed by using the following antibodies: rabbit anti-M 1:1000 (100-401-A55, Rockland TEBU-BIO);rabbit anti-N 1:3000 (200-401-A50,	anti-M suggested: None anti-N suggested: None
   anti-GAPDH antibody coupled to HRP 1:25000 (G9295, Sigma).	anti-GAPDH suggested: (Sigma-Aldrich Cat# G9295, RRID:AB_1078992)
   For immuno-spotting, rabbit anti-Spike neutralizing Antibody 1:100 (40592-R001, Sino Biological), mouse anti-CD81 1:100 (sc7637, 1.3.3.22, Santa Cruz)	anti-Spike suggested: (Sino Biological Cat# 40592-R001, RRID:AB_2857936) anti-CD81 suggested: (Santa Cruz Biotechnology Cat# sc-7637, RRID:AB_627190)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells and culture conditions: The HEK293T human embryonic kidney cell line were obtained from ECACC (Sigma-Aldrich, Germany) and maintained in cultured in Dulbecco’s modified essential medium (DMEM, Gibco) supplemented with 10% heat inactivated fetal calf serum (FCS, Thermo Fisher, USA), 50 U/mL of penicillin (Ozyme, France), 50 μg/mL of streptomycin (Ozyme,	HEK293T suggested: None
   The human pulmonary alveolar A549-hACE2 and A549-hACE2mScarlet stable cell lines were engineered using 2 different lentiviral vectors.	A549-hACE2mScarlet suggested: None
   For A549-hACE2-mScarlet, A549 were transduced with VSVg pseudotypes particules derived from lentiviral vector expressing mScarlet red fluorescent protein from Olivier Schwartz laboratory at Institut Pasteur (Paris, France) to express the human receptor hACE2mScarlet.	A549-hACE2-mScarlet suggested: None
   Transduction efficiency was assessed by monitoring mScarlet expression (Maximum Excitation 569nm/Maximum Emission 594nm) using flow cytometry in comparison with A549 cells (Supplemental Figure 1B).	A549 suggested: None
   Human pulmonary A549-hACE2 cells were harvested live in cold PBS 1x (after 24h in culture on glass coverslip).	A549-hACE2 suggested: RRID:CVCL_A5KB)
   Recombinant DNA
   Sentences	Resources
   The GFP and mEOS2 tags were amplified by PCR from peGFP and pmEOS2 using Pfu Polymerase (M7741, Promega), dNTP mix (10297018, Thermo Scientific) and primers designed to carry NheI and KpnI restriction sites (Sigma Aldrich).	pmEOS2 suggested: None
   PCR amplified inserts were added at the N-terminus of N protein in pcDNA3.1+ plasmid, and purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).	pcDNA3.1+ suggested: RRID:Addgene_117272)
   Software and Algorithms
   Sentences	Resources
   Each band intensity on the immunoblot were quantified using ImagJ software.	ImagJ suggested: None
   Fluorescence intensity fluctuations traces (at least 120) were recorded for 10s and correlated immediately with the built-in Zen software.	Zen suggested: None
   PALM acquisitions were analyzed using the ThunderSTORM plugin in Fiji [23].	Fiji suggested: (Fiji, RRID:SCR_002285)
   13 Images corresponding to 250-300 fluorescent VLPs were analyzed by ImageJ/Fiji software and ComDet Plugin was used for quantifiying the number of fluorescent VLPs and the double GFP/mCherry colocalization.	ImageJ/Fiji suggested: None
   Statistical analyses: Statistical analyses were performed using Origin 8.5 and Graph Pad prism software.	Origin suggested: (Origin, RRID:SCR_014212)
   GraphPad: GraphPad was used for performing graphs.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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2. 

   Version published to 10.1101/2022.03.23.485575 on bioRxiv

   Mar 27, 2022