| Autor: |
David Barrett, Meghan Wyatt, Haim Bar, Mark K. Haynes, Bruce S. Edwards, Larry A. Sklar, Adam Zweifach |
| Jazyk: |
angličtina |
| Rok vydání: |
2022 |
| Předmět: |
|
| Zdroj: |
SLAS Discovery, Vol 27, Iss 5, Pp 314-322 (2022) |
| Druh dokumentu: |
article |
| ISSN: |
2472-5552 |
| DOI: |
10.1016/j.slasd.2022.03.008 |
| Popis: |
The solid tumor microenvironment (TME) suppresses immune responses. Three alterations in the TME converge on a pathway triggered by elevated cyclic AMP (cAMP) that suppresses T cell receptor (TCR) signaling. We developed a phenotypic assay to screen for small molecules that interfere with this pathway using TALL-104 human leukemic cytotoxic T lymphocytes pretreated with prostaglandin E2 to elevate cAMP. Beads coated with anti-CD3 antibodies stimulate lytic granule exocytosis, which is detected via binding of an antibody against lysosome associated membrane protein 1 (LAMP-1) measured with flow cytometry. Confirming that the assay can find compounds with desired activity, treating cells with a phorbol ester restores exocytosis. The assay behaves well in 96-well format and we screened a collection of compounds expected to have effects on epigenetic regulatory proteins. Compounds in this collection affected lytic granule exocytosis after 24-hour treatment, but none prevented cAMP from suppressing lytic granule exocytosis. We used a fully automated 384-well version of the assay to screen the Prestwick Compound Library but obtained no confirmed hits. Analyzing this assay's performance reveals two points of interest. First, cytometry offers multiple ways to quantify signals. Z’ was higher using percent positive cells than mean fluorescence because the relationship between the two measures saturates, but using percent positive could make it harder to find hits in some assays. Second, variance was higher in positive controls than in negative controls in this assay, which degrades assay performance less than if variance was higher in negative controls. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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