Lactate dehydrogenase-release assay: A reliable, nonradioactive technique for analysis of cytotoxic lymphocyte-mediated lytic activity against blasts from acute myelocytic leukemia

Abstract

Treatment of patients in remission of acute myelocytic leukemia using immunotherapy with interleukin 2 is a new approach to prolonging remission duration in this disease. As an important mechanism for the pathophysiology of eradication of residual myelocytic blast populations, activation of cytotoxic effector lymphocytes has frequently been discussed. However, the associated immunological research has been complicated to some extent, because in conventional chromium 51-release assays, blast cells frequently fail to incorporate sufficient amounts of 51Cr and/or spontaneously release high amounts of 51Cr. Recently, we established a culture system which promotes the outgrowth of cytotoxic T lymphocytes in bone marrow-derived mononuclear cells cultured in IL-2. To study cytotoxicity and the responsible mechanisms of the obtained T-cell lines and clones, we modified a previously described cytotoxicity assay, based on the release of lactate dehydrogenase (LDH-release assay) for use in cryopreserved blasts obtained from the bone marrow of patients with acute myelocytic leukemia. Using this assay, we were able to detect cytotoxicity of IL-2-activated peripheral blood lymphocytes from three healthy controls against a number of blast samples obtained from the bone marrow of patients with AML (up to more than 40% lysis at an effector target cell ratio of 20:1). However, a minority of AML blasts seem to be resistant to lysis by IL-2-activated lymphocytes. In bone marrow-derived T-cell lines from patients with AML we detected lytic activity against autologous blasts in three of seven cases tested by LDH release, ranging from 29 to 63% at an effector target ratio of 10:1. Additionally, T-cell clones with different phenotypes were established which were able to mediate cytotoxicity against autologous blast cells. Thus, cytotoxicity against freshly isolated blasts from patients with acute myelocytic leukemia can be analyzed reliably, reproducibly, and without the use of isotopes by the LDH-release assay.

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl-d-aspartate receptors in cultured rat hippocampal neurons

“…The activity of LDH released into culture medium was determined by the enzyme-coupled color development method as previously described (Weidmann et al, 1995) with minor modifications. In brief, cells were exposed to NMDA or other compounds, and further cultured for an additional 24 h in DMEM without phenol red.…”

Effects of Naproxen on Immune Responses in a Colchicine-Induced Rat Model of Alzheimer's Disease

“…Cytotoxicity of splenic MNC (the effector cells collected from the spleen using the procedure described for LAI) against target cells (EAC cells) was tested in a lactate dehydrogenase (LDH) release assay using the LDH-FS non-radioactive cytotoxicity assay kit (DiaSys Diagnostic Systems GmbH, Germany) following the procedure of Weidmann et al [40]. Effector as well as target cells were washed separately in 15-ml centrifuge tubes with PBS and their pellets were dissolved separately in phenol red-free RPMI 1640 (HiMedia) medium.…”

The preparation of leukemia cell vaccine expressing BCG heat shock protein 70 and anti-leukemia effect in vitro

“…In this study, (1) the proliferation indices of T cells were assayed by CFSE-staining method with flow cytometry [32,33], which showed that the BCG HSP70 gene transfected AML/B-ALL cells significantly enhanced lymphocyte proliferation; (2) cytokine level by ELISA suggested that HSP70-transfected AML/B-ALL cells stimulated autologous T cells to secrete the highest cytokine level (IFN-γ); (3) lactate dehydrogenase (LDH) release assay used to measure killing activity of CTLs [34] showed that the killing rate of CTL in the group of HSP70-transfected AML/B-ALL cells was significantly higher than that of the wide-type AML/B-ALL cells and pDisplay-transfected ones, suggesting that BCG HSP70 gene transfection could significantly enhance the killing activity of autologous CTL on leukemia cells.…”