Authentication Reports  

The authentication plan for the cell lines is based on the International Cell Line Authentication Committee (ICLAC)'s "Cell Line Checklist for Manuscripts and Grant Applications"(1).

1. Check dkNET for Misidentified Cell Lines

   If you have not created a dkNET Authentication Report, you can check for this information via dkNET Resource Reports.
   Check Resource Information

   To verify that this is not a false cell line, misidentified, or to check this is known to be an authentic stock, please check via dkNET. dkNET obtains this information from Cellosaurus (2).

   
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2. Provide the following information in the Authentication of key biological and/or chemical resources attachment(3) in the grant application:

   1. Identification of cell lines

      1. Provide Name, Vendor, Catalog#, RRID (Find RRID via dkNET Resource Report)

      2. If you can't find your cell lines in the system, please register it at Cellosaurus(2). An RRID will be generated in 1-2 business day.

   2. Short Tandem Repeat (STR) Profiling

      The gold standard for authentication testing of cell lines is STR profiling. STR profiling should be performed and compared to results from donor tissue, or to online databases of cell line STR profiles. Authentication testing should be performed on established cell lines regardless of the application, and the test method and results included in the Materials and Methods section of any research publication. Testing should be done, at minimum, at the beginning and end of experimental work(4, 5).

Sources:

1. Cell Line Checklist for Manuscripts and Grant Applications, International Cell Line Authentication Committee (ICLAC), version 1.2, updated on May 9, 2014.
2. Cellosaurus (https://web.expasy.org/cellosaurus/) is a cell line knowledge resource and nomenclature authority. Cellosaurus covers the information of "ICLAC register of misidentified cell lines" (latest version 8.0, released Dec. 1, 2016,) and provides more updated information.
3. Guidance: Rigor and Reproducibility in Grant Applications
4. Standards for Cell Line Authentication and Beyond, The National Institute of Standards and Technology (NIST).
5. Published standard: ANSI/ATCC ASN-0002-2011, Authentication of Human Cell Lines: Standardization of STR Profiling, American National Standards Institute (ANSI).

This authentication plan for antibodies is based on the methods suggested in "A proposal for validation of antibodies" (Uhlen M et. al., 2016)(1), the guideline published in the Journal of Comparative Neurology (Saper C, 2005)(2), and the Example Authentication of of Key Biological and/or Chemical Resources (Bandrowski A)(3).

1. Check dkNET for Known Issues in Antibodies

   If you have not created dkNET Authentication Reports, you can check the following information at dkNET Resource Reports.
   Check Resource Information

   Please check your antibody information at dkNET to determine whether there are any known issue. dkNET obtains this information from the Antibody Registry(4).

2. Provide the following information in the Authentication of key biological and/or chemical resources attachment(5) in the grant application:

   1. Identification of antibodies

      1. Provide Name, Vendor, Catalog#, RRID (Find RRID via dkNET Resource Report)

      2. If you can't find your antibody in the system, please register it at Antibody Registry(4). An RRID will be generated in 1-2 business day.

   2. Validation

      1. Antibody validation must be carried out in an application- and context-specific manner (Uhlen et al., 2016), i.e., just because you used an antibody successfully in one application, doesn’t mean it will translate. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used (Bordeaux et al., 2014). See ii. Suggested validation methods. When selecting an antibody to use, pay special attention to the following:
         Check Resource Information

         1. Check Target Organism - Check to see if the antibody has been developed for and tested in the target species for your experiment in the Target Organism field.

         2. Check Application - Check if your planned applications included in the recommended applications provided by the vendor listed in the Comments field. We do not recommend using an antibody that has not been tested for a specific application.

         3. Check Validation Information - Check validation information to see if it is a widely used reagent or if any concerns have been raised.

      
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      2. Suggested validation methods based on applications(1)
   
   

   Validation strategy	Genetic	Orthogonal	Independent antibody	Tagged protein expression	IMS
   Validation principle	The expression of the target protein is eliminated or significantly reduced by genome editing or RNA interference	Expression of the target protein is compared with an antibody-independent method	Expression of the target protein is compared using two antibodies with nonoverlapping epitopes	The target protein is expressed using a tag, preferably expressed at endogenous levels	The target protein is captured using an antibody and analyzed using MS
   Validation criteria	Elimination or significant reduction in antibody labeling after gene disruption or mRNA knockdown Search dkNET for knockout organism	Significant correlation of protein levels detected by an antibody and an orthogonal method (e.g., MS)	Significant correlation of protein levels detected by two different antibodies recognizing independent regions of the same target protein	Significant correlation between antibody labeling and detection of the epitope tag	Target protein peptides among the most abundant detected by MS following immunocapture
   Suitable for these applications	WB, IHC, ICC, FS, SA, IP/ChIP, RP	WB, IHC, ICC, FS, SA, RP	WB, IHC, ICC, FS, SA, IP/ChIP, RP	WB, IHC, ICC, FS	IP/ChIP

   WB, western blot; IHC, immunohistochemistry; ICC, immunocytochemistry, including immunofluorescence microscopy; FS, flow sorting and analysis of cells; SA, sandwich assays, including ELISA; IP, immunoprecipitation; ChIP, chromatin immunoprecipitation; and RP, reverse-phase protein arrays.

Sources:

1. Uhlen M et. al. A proposal for validation of antibodies. Nature Methods, Oct;13(10):823-7. 2016.
2. Saper C. An open letter to our readers on the use of antibodies. Journal of comparative neurology, 493(4):477-8, 2005.
3. Bandrowski A. Example Authentication of of Key Biological and/or Chemical Resources, NIH Policy on Rigor and Reproducibility Section, UC San Diego Library website.
4. AntibodyRegistry (https://antibodyregistry.org)
5. Guidance: Rigor and Reproducibility in Grant Applications, National Institute of Health Office of Extramural Research Website.