Intracellular Flow Cytometry Complements RT-qPCR Detection of Circulating SARS-CoV-2 Variants of Concern

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1. Version published to 10.2144/btn-2022-0018

   Apr 21, 2022
2. 

   ScreenIT

   Feb 5, 2022

   SciScore for 10.1101/2022.02.02.478775: (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	Contamination: 2.1 Cell lines and virus strains: Cell lines: African green monkey kidney cells (Vero E6 cells) were obtained from ATCC (CRL-1586) (Manassas, VA, USA) as mycoplasma-free stocks and were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific (TFS), Merelbeke, Belgium) supplemented with 10 % fetal bovine serum (FBS), 2 mM L-glutamine (TFS) and 0.075 % sodium-bicarbonate (TFS).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   goat anti-Rabbit IgG monoclonal antibody was from Cell Signaling Technologies (MA, USA; …

   SciScore for 10.1101/2022.02.02.478775: (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	Contamination: 2.1 Cell lines and virus strains: Cell lines: African green monkey kidney cells (Vero E6 cells) were obtained from ATCC (CRL-1586) (Manassas, VA, USA) as mycoplasma-free stocks and were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific (TFS), Merelbeke, Belgium) supplemented with 10 % fetal bovine serum (FBS), 2 mM L-glutamine (TFS) and 0.075 % sodium-bicarbonate (TFS).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   goat anti-Rabbit IgG monoclonal antibody was from Cell Signaling Technologies (MA, USA; Cat. n° 4414).	goat anti-Rabbit IgG monoclonal antibody suggested: None anti-Rabbit IgG suggested: (Cell Signaling Technology Cat# 4414, RRID:AB_10693544)
   Samples were washed twice with Perm/Wash buffer before the addition of the primary (anti-Nucleocapsid) antibody (0.3 μg per sample).	anti-Nucleocapsid suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   To determine replication efficiency, the different SARS-CoV-2 strains were added to Vero E6 cells at an MOI of 0.02, as determined by end-point dilution titration on Vero E6 cells and calculated by the tissue culture infectious dose 50 (TCID50) method of Reed and Muench (5).	Vero E6 suggested: None
   Software and Algorithms
   Sentences	Resources
   Acquisition of all samples was done on a BD FACSCelesta flow cytometer (BD Biosciences) with BD FACSDiva v8.0.1 software.	BD FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
   Flow cytometric data were analyzed in FlowJo v10.1 (Tree Star).	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • 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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3. 

   Version published to 10.1101/2022.02.02.478775 on bioRxiv

   Feb 2, 2022