This protocol is for the detection of cytokine production by activated antigen-specific T cells in vitro. If other stimulants or stimulated cells are used in vitro (e.g. monocytes stimulated with L P S), or if in vivo stimulated cells are analyzed directly in vitro, start by labeling the stimulated cells with a capture reagent (step 6 ). Author: J.E. Colligan et al. Translated by Xuitao Cao et al. This experiment is from the "Compendium Immunology Laboratory Guide".
Operation method
Detection of cytokine secretion and isolation by flow cytometry Cells secreting cytokines Move Basic program cytokine secretion assay Materials Ice-cold or 37°C pre-warmed culture medium. For example, human cells are cultured in 1640 complete culture medium containing 10 % human AB plasma or own serum. For culturing mouse cells, use RPM 1-1640 complete medium with 10 % mouse serum. Antigen, 1 to 10ug/m l peptide or 1 to 100ug/m l protein Suitable control antigen Staphylococcus aureus enterotoxin (S E B, Sigma) P B S , p H 7. 2 , containing 0.5% (m /V ) B S A and 2 mmo l /L E D T A , pre-cooled on ice Cytokine capture reagent: primary antibody, containing antibodies against cell surface antigens (e.g. CD 45) and anti-cytokine antibodies (Miltenyi Biotec) Recombinant cytokines Antibodies for cytokine detection (Miltenyi Biotec): P E, A PC (allophycocyanin) or FITC-labeled anti-cytokine monoclonal antibodies Fluorescein-labeled antibodies: anti-human or mouse CD 4-FITC, anti-human CD M-PerCP, anti-mouse CD 45R/B 220-PerCP, etc. Anti-PE beads (Miltenyi Biotec) 9 6-well or 2 4-well plates or 6cm dishes Cell scraper or similar tool 1.5 to 15 ml v-bottom tubes or deep-well culture plates Slow rotating incubator 1 . Preparation of human P B M C (Module 8 . 1 ) or mouse spleen/lymph node cells (Module 2.1). Counting of cells and determination of cell activity Do not use any non-human (human cells) or non-mouse (mouse cells) in cell isolation, stimulation and freezing. 1 . Prepare and wash the cells (see basic protocol, steps 1 and 2). Suspend the cells in culture medium: I X l O7 cells/ml. 2 . Add IOOul of cell suspension containing IX IO6 cells to each 1.5 ml V-bottom tube or deep-well culture plate. Label as A 3 . Add antigen and incubate cells as in step 4 of the basic protocol. For more product details, please visit Aladdin Scientific website.
