An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells
Published: November 30, 2023
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. 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.</l…
Divulgaciones
The authors have nothing to disclose.
Materials
| 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. |
Citar este artículo
An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells. J. Vis. Exp. (Pending Publication), e21838, doi: (2023).