Determination of Presence of ADAMTS7 in Several Human Cell Lines

Songjukta Chakraborty, Joshua Muia, Anna Platt, Bhuvanesh Kumar Raju, Frida Miranda, Thomas Momanyi, Ayomide Ishola

Research output: Contribution to conferencePosterpeer-review

Abstract

Background: According to studies, ADAMTS7 is associated with coronary atherosclerotic diseases. To understand the regulation of ADAMTS7, we have cultured the following laboratory available human cell lines to 3 or 4 passages: Hela, Human gingival fibroblast (HGF-1), Liver hepatocellular carcinoma (HepG2), and Human umbilical vein endothelial cells (HUVEC). These passage numbers are important because genetic and phenotypic changes will be less during early passages. After having 70 to 80% confluency, media was changed to Freestyle media and harvested after 48 hours of conditioning to do Western blotting. During the Western blot technique, primary antibody will bind with ADAMTS7 if this enzyme is present in the specific cell culture media. This process will help to understand the cell type and the amount of ADAMTS7 present in the cells.

Method: We took the cell lines such as Hela, Human gingival fibroblast (HGF-1), Liver hepatocellular carcinoma (HepG2), Human umbilical vein endothelial cells (HUVEC) from cryogenic liquid and grew into T-25 flasks in DMEM or F-12K media with additives respectively. After subculturing 3 to 4 passages into the T-150 flask, we replaced the media with Freestyle media when the cells are 70 to 80% confluent. We harvested the media, added biotin to 10ml, and took samples of the biotinylated and non-biotinylated media for Western blotting. Then 25 ml conditioned media was concentrated by using viva spin columns and a sample of the concentrated media was taken for Western blotting.

Result & Conclusion: Western blotting with the concentrated, unconcentrated and biotinylated media to determine the expression of ADAMTS7 in specific human cell lines. Additionally, we will study that which cell lines are appropriate for ADAMTS7. We will further study the localization of ADAMTS7 by using imaging and microscopy.
Original languageAmerican English
Pages24
StatePublished - 17 Feb 2023
EventOklahoma State University Center for Health Sciences Research Week 2023 - Oklahoma State University Center for Health Sciences, 1111 W. 17th street, Tulsa, United States
Duration: 13 Feb 202317 Feb 2023
https://medicine.okstate.edu/events/index.html?trumbaEmbed=view%3Devent%26eventid%3D160681489

Conference

ConferenceOklahoma State University Center for Health Sciences Research Week 2023
Country/TerritoryUnited States
CityTulsa
Period13/02/2317/02/23
Internet address

Keywords

  • atherosclerosis
  • Western Blotting
  • biotin
  • human cell culture

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