An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells

Abstract

Source: Hodge, M. X., et al. In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure. J. Vis. Exp. (2019).

This video demonstrates a method to induce apoptosis in the Jurkat cells — a human T lymphocyte leukemia cell line. The cells are exposed to ultraviolet C (UVC) radiation, which causes extensive DNA damage and leads to apoptotic cell death.

Protocol

1. Preparation of Jurkat T cell line (Day 2)

NOTE: All procedures should be conducted in a class II biological safety cabinet.

1. Culture Jurkat T cells in 24 mL of basal cell culture medium + 10% FBS + 5% penicillin/streptomycin at 37 °C + 5% CO₂ (Table of Material). Jurkat T cells are a suspension cell line that can be maintained through passaging 1:6-1:8 into pre-warmed culturing media every 3 days. Do not shake.
2. To prepare apoptotic cells, grow them to 90% confluency in each flask (which takes 3-4 days to achieve after passaging). For this study, use c....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Annexin V-FITC Kit	Trevigen	4830-250-K	The TACS Annexin V-FITC Kit allows rapid, specific, and quantitative identification of apoptosis in individual cells when using flow cytometry.
BCL2 Jurkat T Cells	ATCC	ATCC CRL-2899	The BCL2 Jurkat cell line was derived by transfecting human Jurkat T cells with the pSFFV-neo mammalian expression vector containing the human BCL-2 ORF insert and a neomycin-resistant gene. Has been for models of measuring efferocytosis.
Countess II Automated Cell Counter	Thermofisher	AMQAX1000	It is a benchtop assay platform equipped with state-of-the-art optics, full autofocus, and image analysis software for rapid assessment of cells in suspension. Very easy to use.
Cytospin 4 Cytocentrifuge	Thermofisher	A78300003	Provides economical thin-layer preparations from any liquid matrix, especially hypocellular fluids such as bronchoalveolar lavage fluid.
Fetal Bovine Serum, qualified, heat inactivated	Thermofisher	16140071	Provides Nutrients to cultured cells for them to grow. It is standard for cell culture.
Penicillin-Streptomycin	Sigma/Aldrich	P0781-100ML	Penicillin-Streptomycin is the most commonly used antibiotic solution for culture of mammalian cells. Additionally it is used to maintain sterile conditions during cell culture.
RPMI 1640 Medium, GlutaMAX Supplement	Thermofisher	61870036	RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium) was originally developed to culture human leukemic cells in suspension and as a monolayer. RPMI 1640 medium has since been found suitable for a variety of mammalian cells, including HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes, and carcinomas. Helps grow Jurkat T cells fast and efficiently.
Stratagene UV Stratalinker 1800 UV Crosslinker	Cambridge Scientific	16659	The Stratalinker UV crosslinker is designed to induce apoptosis, crosslink DNA or RNA to nylon, nitrocellulose, or nylon-reinforced nitrocellulose membranes.