Longping Victor Tse University of North Carolina at Chapel Hill

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Garrett Berry Gene Therapy Center, The University of North Carolina at Chapel Hill, USA
1 protocol

Gary Whittaker Department of Microbiology and Immunology, Cornell University, Ithaca NY
2 protocols

Yueting Zhang Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, USA
1 protocol

Akira Karasawa New York Structural Biology Center
10 protocols

Arsalan Daudi University of California
157 protocols

Beatrice Li University of Oxford
7 protocols

Christina Tan A*STAR Skin Research Lab
4 protocols

Editor

Longping Victor Tse

Post-Doc, University of North Carolina at Chapel Hill

Research focus

Gene Therapy
Gene Delivery
Vector Engineering
Infectious Diseases

2 Author merit
Reviewer merit
Editor merit

Education

PhD, Cornell University, 2014

Publications

https://scholar.google.com/citations?user=zC4XSPIAAAAJ&hl=en

2 Protocols published

Virus Binding and Internalization Assay for Adeno-associated Virus

Authors: Garrett E. Berry and Longping V. Tse, date: 01/20/2017, view: 10609, Q&A: 0

The binding and internalization of adeno-associated virus (AAV) is an important determinant of viral infectivity and tropism. The ability to dissect these two tightly connected cellular processes would allow better understanding and provide insight ...

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Immunoplaque Assay (Influenza Virus)

Authors: Longping V. Tse, Yueting Zhang and Gary R. Whittaker, date: 11/05/2013, view: 22928, Q&A: 1

Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the ...

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Ramuth Magalutcheemee

Hello ,
Thank-you very much for sharing the Immunoplaque assay protocol .
In Fact the plaque assay did not work due to some constraint from our part :
1. We did not have the 6-well tissue culture dish but used individual tissue culture plate which we placed on a tray to giggle the plate to evenly distribute the media.
2. The cell monolayer (MDCK cell line ) was not 90 % confluent , so we incubated the plate for 2-3 days . Finally the cells grew but not around the rim of the plate. (See picture)
3. Even so we proceeded with inoculation of virus dilutions (Influenza virus) and overlaid the plate with Noble agar , instead of agarose .
4. We did not have MTT available for staining but used 1% Crystal violet .
Final result : No plaque visible .
Quite discouraging but will try again !!

12/13/2018 6:37:35 PM Reply

Longping Tse

University of North Carolina at Chapel Hill

Hi Ramuth,

I will suggest you to keep the MDCK cells at 90% when you start your inoculation. Fifty percent is too low for plaque assay. Good Luck!

Cheers,
Vic

2019-01-15 07:01:04

Ramuth Magalutcheemee

Hello Vic ,
Thank-you for the valuable advice.
In fact i did not fix the cell monolayer , I will do so next time .
Confluency also i used 80 % . Will use 50 % in future plaque assay .
Thanks again .

Kind Regards ,
Vanessa

2018-12-14 07:43:25

Longping Tse

University of North Carolina at Chapel Hill

Dear Ramuth,

I am sorry that your plaque assay did not work, but you are definitely not the first one. Plaque assay is quite tricky to deal with. I put some pointers below and hope it will help.

1. There are multiple type of MDCK cells, MDCK.1, MDCK.2 and the generic MDCK which is a mix of the two. For more information, please refer to the paper below.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209442/

2. Do not let your MDCK go to 100%, since plaque assay tend to take at least 2 -3 days to finish after the inoculation of the viruses. Your MDCK might overgrown and died if your initial confluence is too high. Judging from the picture you provided, your MDCK cells died or detached before your staining.

3. Did you fix the cell mono-layer with 4% PFA? A lot of people tend to fix the cells after the removal of the agar, I personally found fixing it before removing the agar help to better retain the mono-layer structure.

4. My protocol have another twist from the generic protocol, I basically did immunohistology instead of crystal violet to visualize the plaques. It woks exceptionally well for tiny plaque.

Please let me know if you have further questions, I will be glad to help.

Cheers,
Vic

2018-12-14 06:14:46

5 Protocols reviewed

Expression and Purification of the Cas10-Csm Complex from Staphylococci

Authors: Lucy Chou-Zheng and Asma Hatoum-Aslan, date: 06/05/2017, view: 9646, Q&A: 0

CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein ...

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Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors

Authors: Chin-kai Chuang, Ching-Fu Tu and Chien-Hong Chen, date: 06/05/2017, view: 10084, Q&A: 0

A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription ...

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23 Protocols edited

VirScan: High-throughput Profiling of Antiviral Antibody Epitopes

Authors: Ellen L. Shrock, Christine L. Shrock and Stephen J. Elledge, date: 07/05/2022, view: 33, Q&A: 0

Profiling the specificities of antibodies can reveal a wealth of information about humoral immune responses and the antigens they target. Here, we present a protocol for VirScan, an application of the phage immunoprecipitation sequencing (PhIP-Seq)

...

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Environmental Conditioning and Aerosol Infection of Mice

Authors: Eriko Kudo and Akiko Iwasaki, date: 04/20/2020, view: 3311, Q&A: 0

Influenza infection models in mice are widely used to study flu-mediated immune responses and pathology. However, most laboratory mice are housed at 20 °C and 50% relative humidity (RH). To better recapitulate influenza epidemics and immune ...

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