A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

被引：2

作者：

Chew, Katherine ^{[1
]}

Lee, Branden ^{[1
]}

Ozonoff, Al ^{[1
,2
]}

Smith, Jennifer A. ^{[3
]}

Levy, Ofer ^{[1
,2
,4
]}

Dowling, David J. ^{[1
,2
]}

Van Haren, Simon ^{[1
,2
]}

机构：

[1] Boston Childrens Hosp, Precis Vaccines Program, Div Infect Dis, Boston, MA 02115 USA

[2] Harvard Med Sch, Dept Pediat, Boston, MA 02115 USA

[3] Harvard Med Sch, ICCB Longwood Screening Facil, Boston, MA USA

[4] Broad Inst MIT & Harvard, Cambridge, MA USA

来源：

STAR PROTOCOLS | 2023年 / 4卷 / 03期

基金：

美国国家卫生研究院;

关键词：

Biotechnology and bioengineering; Cell-based Assays; Flow Cytometry/Mass Cytometry; High-Throughput Screening; Immunology;

D O I：

10.1016/j.xpro.2023.102405

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

High-throughput screening is a powerful platform that can rapidly provide valu-able cytotoxic, immunological, and phenotypical information for thousands of compounds. Human peripheral blood mononuclear cells (PBMCs) cultured in autologous plasma can model the human immune response. Here, we describe a protocol to stimulate PBMCs for 72 h and measure cytokine secretion via AlphaLISA assays and cell surface activation marker expression via flow cytome-try. Cryopreserved PBMCs are incubated for 72 h with various small molecule li-braries and the supernatants are harvested to rapidly measure secretion levels of key cytokines (tumor necrosis factor alpha, interferon gamma, interleukin 10) via the AlphaLISA assay. Almost simultaneously, the cells can be fixated and stained using antibodies against innate immune activation markers (CD80, CD86, HLA-DR, OX40) for analysis via flow cytometry. This multiplexed readout workflow can directly aid in the phenotypic identification and discovery of novel immuno-modulators and potential vaccine adjuvant candidates. For complete details on the use and execution of this protocol, please refer to Chew et al.1

引用

页数：15

共 50 条

[31] High-throughput screening
Wallace, RW
DRUG DISCOVERY TODAY, 1998, 3 (02) : 92 - 93
[32] High-throughput screening
Persidis, A
NATURE BIOTECHNOLOGY, 1998, 16 (05) : 488 - 489
[33] High-throughput screening
不详
GENETIC ENGINEERING & BIOTECHNOLOGY NEWS, 2007, 27 (20): : 1 - +
[34] High-throughput screening
Rogers, MV
DRUG DISCOVERY TODAY, 1997, 2 (06) : 251 - 251
[35] High-throughput screening
Rogers, MV
DRUG DISCOVERY TODAY, 1997, 2 (05) : 209 - 209
[36] High-throughput screening
Nature Biotechnology, 2001, 19 (7) : 689 - 689
[37] High-throughput screening
Nature Biotechnology, 2005, 23 (6) : 755 - 755
[38] A high-throughput screening protocol for fast evaluation of enantioselective catalysts
Wolf, C
Hawes, PA
JOURNAL OF ORGANIC CHEMISTRY, 2002, 67 (08): : 2727 - 2729
[39] High-Throughput Screening
Wildey, Mary Jo
Haunso, Anders
Tudor, Matthew
Webb, Maria
Connick, Jonathan H.
ANNUAL REPORTS IN MEDICINAL CHEMISTRY, VOL 50: PLATFORM TECHNOLOGIES IN DRUG DISCOVERY AND VALIDATION, 2017, 50 : 149 - 195
[40] High-throughput screening
Nature, 2001, 413 (6855) : 549 - 550

← 1 2 3 4 5 →