A novel cytotoxic T lymphocyte activation assay: Optimized conditions for antigen receptor triggered granule enzyme secretion
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The NKG2D ligand ULBP4 binds to TCRγ9/δ2 and induces cytotoxicity to tumor cells through both TCRγδ and NKG2D
2009, BloodCitation Excerpt :After 48 hours, cytokine in cell-free supernatants was determined by ELISA. For measurement of granule release, cells were stimulated for 5 hours at 37°C and supernatants were tested with a standard N-benzyloxycarbonyl lysine thiobenzyl ester (BLT3; Sigma-Aldrich) esterase assay.16,33 All assays were performed in triplicate wells, and results are presented as the mean from the given culture conditions.
Calcineurin activation is only one calcium-dependent step in cytotoxic T lymphocyte granule exocytosis
2007, Journal of Biological ChemistryCitation Excerpt :Ca2+-free Ringer's solution contained 145 NaCl, 4.5 KCl, 1 MgCl2, 1 mm EGTA, 5 HEPES, and 10 glucose (pH 7.4 with NaOH). Nα-Benzyloxycarbonyl-l-lysine Thiobenzyl ester (BLT) Esterase Assays—Granyzme B released from TALL-104 cells was assayed by measuring hydrolysis of BLT essentially as described previously (13, 20, 22). Absorbance measurements were made with a Bio-Tek Synergy HT-I plate reader (Bio-Tek Instruments, Winooski, VT) read at 410 nm after subtraction of an appropriate blank.
Uncoupling of T-cell effector functions by inhibitory killer immunoglobulin-like receptors
2006, BloodCitation Excerpt :Standard 4-hour 51Cr-release or CytoTox 96 Non-Radioactive Assays (Promega, Madison, WI) were carried out with P815 cells coated with mouse IgG, anti-CD3 and mouse IgG, or anti-CD3 and anti-KIR2DL2 mAbs as target cells, and either KIR2DL3+ NK cells or CD4+CD28–KIR2DL2+ T cells as effector cells. BLT esterase assays were used to assess degranulation.19 Effector cells were incubated with either antibody-coated P815 cells or with SEB-coated 721.221 or 721.221/HLA-Cw3 target cells; supernatants were collected after 4 hours.
Cross-talk with Ca<sup>2+</sup> influx does not underlie the role of extracellular signal-regulated kinases in cytotoxic T lymphocyte lytic granule exocytosis
2004, Journal of Biological ChemistryCitation Excerpt :Data were analyzed further using Igor Pro software (Wavemetrics, Lake Oswego, OR). Granzyme Release Assays—Granyzme released from TALL-104 cells was assayed by measuring hydrolysis of Nα-benzyloxycarbonyl-l-lysine thiobenzyl ester (BLT) essentially as described previously (36). Tall-104 cells were suspended at a density of 5 × 107 cells/ml of cell culture medium.
A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes
2004, Journal of Immunological MethodsImmune reactions
2003, NeuroImmune Biology