Cytotoxicity was measured by a 4 hr chromium release assay as previously described [63 (link)]. Experiments were performed in triplicate. Data are expressed as the percentage of specific 51Cr release from target cells, calculated as (experimental release-spontaneous release)/(maximum release-spontaneous release) × 100. In some experiments, the inhibition of the PFN/Gzm-mediated cytotoxic pathway was performed by using NK effector cells pre-incubation for 2 hr with 100 nM concanamycin A (CMA)(Sigma-Aldrich) (concentration of CMA during lysis: 50 nM). The inhibition of the NKG2D-dependent cytotoxic was performed by using NK effector cells pre-incubation for 30 min with 0.5 μg/mL anti-NKG2D blocking mAb (clone Bat-221; Miltenyi Biotec.) which was maintained at the same concentration during lysis.
Concanamycin a cma
Manufactured by Merck Group
Sourced in United States
Concanamycin A (CMA) is a laboratory compound produced by Merck Group. It is a macrolide antibiotic that functions as a potent and specific inhibitor of vacuolar-type H+-ATPases (V-ATPases). V-ATPases play a crucial role in regulating intracellular pH and membrane trafficking processes.
Automatically generated - may contain errors
Lab products found in correlation
Mevastatin, by Merck Group (1 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Z vad fmk, by BD (1 mentions)
W6 32 or l243 monoclonal antibody, by BioLegend (1 mentions)
Ldh release assay kit, by Promega (1 mentions)
Human cd56 microbeads, by Miltenyi Biotec (1 mentions)
Gamma counter, by PerkinElmer (1 mentions)
3 4 dichloroisocoumarin dci, by Merck Group (1 mentions)
7 protocols using concanamycin a cma
1
Cytotoxicity Assay for NK Cells
2
Cytotoxicity of Expanded γδ T Cells on Osteosarcoma
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The cytotoxicity of human ex vivo expanded γδ T cells on osteosarcoma cells was evaluated by MTS assay. Osteosarcoma cell lines as well as primary tumor cells were seeded in 96-well plates at 3–5 × 103 cells/well. After 24 h, they were pre-treated with VPA (Sigma) or/and ZOL at indicated concentrations for 24 h before co-cultured with γδ T cells from healthy volunteers or osteosarcoma patients at various E:T ratios. After co-culture for 2 h at 37°C, the supernatant was removed and the wells were softly washed with PBS twice. Then the cytotoxic effect was measured by MTS assay following the manufacturer’s instructions. A MR7000 microplate reader (Dynatech, NV, USA) was used to quantify the percentage of survived cells by determining the optical density. To inhibit mevalonate intermediates-mediated recognition by γδ T cells, osteosarcoma cells were treated with Mevastatin (Sigma) at 5 µM 1 h prior to treatment with VPA or/and ZOL for 24 h. Mevastatin was re-added at time of co-culture to maintain a constant concentration. To inhibit perforin-mediated cytotoxicity, γδ T cells were incubated with concanamycin A (CMA, Sigma) at 100 ng/ml for 2 h at 37°C before co-culture. To block the relevant cytotoxic pathways, specific mAbs were used at 10 mg/ml just before co-incubation assay.
3
Chromium Release Assay for Cytotoxic T-Cell Activity
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Primary human islets or βL5 cells were used as target cells in the chromium release assays (CML) as previously described (36 (link)). Human islets from donors with HLA-A*02 were selected. Prior to plating, human islets were washed with PBS and dispersed in cell dissociation buffer (Life Technologies) for 10 min at 37°C with gentle pipetting every 3 min. Dispersed islet cells were plated at 40,000 cells/well. βL5 cells express IGRP in the context of HLA-A*0201 and were seeded at 10,000 cells/well in 96-well flat bottom plates. CML was performed as described previously using IGRP-CTL avatars as effectors (37 (link)). Specific lysis was calculated as follows: [(Experimental Release − Spontaneous Release)/(Maximum Release − Spontaneous Release)] × 100. For inhibition of granule exocytosis, CTLs were incubated with 100 nmol/L of concanamycin A (CMA) (Sigma-Aldrich) for 2 h. For inhibition of Fas ligand (FasL)-mediated killing or caspase activation, 0.5 µg/mL of anti-Fas blocking antibody or 50 µmol/L of the pan-caspase inhibitor, Z-VAD-FMK (BD Biosciences), respectively, was added to target cells 2 h prior to coculture with CTLs.
4
Cytotoxicity Assay for SP17-Stimulated T Cells
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A standard 4-hour LDH release cytotoxicity assay (LDH Release Assay Kit, Promega, USA) was performed to evaluate the cytotoxic activity of the SP17-stimulated T cells. Cytotoxicity against autologous breast cancer primary cells was determined at various effector:target cell ratios. For the measurement of CTL-mediated lysis of autologous lymphoblastoid cells (LCL) pulsed with SP17 or HPV16-E6 antigen, cytotoxicity assay was performed with 20:1 effector:target ratio. LCL were generated from autologous PBMCs as described previously [26 (link)]. To determine HLA restricted response, a cytotoxicity assay was performed with or without 25 μg/mL HLA-I or HLA-II (W6/32 or L243 monoclonal antibody, respectively, BioLegend 11080 Roselle Street, San Diego, CA) blocking antibodies (effector:target ratio 20:1). To block the perforin-mediated cell lysis, 5 μg/mL concanamycin A (CMA, Sigma-Aldrich, USA) was added to the co-cultures [27 (link)].
5
NK Cell-Mediated Cytotoxicity Assay
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Peripheral blood from healthy donors was obtained from the NIH Blood Bank (Bethesda, MD) under the appropriate Institutional Review Board approval and informed consent. NK cells were isolated using human CD56+ MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Target cells were labeled with 111In oxide and seeded in triplicate at 3.0 × 103 cells/well in 96-well round-bottom culture plates with effector NK cells at various effector-to-target (E:T) ratios. Following 16 h incubation, supernatants were harvested and the release of 111In was measured utilizing a gamma counter (PerkinElmer, Waltham, MA). Spontaneous release was determined by incubating the target cells with medium alone, and complete lysis was determined by incubating the target cells with 1% triton X-100 in water. Specific lysis was calculated as follows: % specific lysis = [(observed release – spontaneous release) / (complete release – spontaneous release)] × 100. To inhibit the function of perforin/granzyme, NK cells were preincubated with 200 nM concanamycin A (CMA, Sigma Aldrich) for 2 h at 37°C.
6
NK Cell Cytotoxicity Assay Protocol
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NK cell cytotoxic activity was measured by a 4 h 51Cr release assay performed in triplicate, as previously described27 (link). Data are expressed as the percentage of specific 51Cr release from target cells, calculated as (experimental release-spontaneous release)/(maximum release-spontaneous release) × 100. Inhibition of the PFN/Gzm-mediated cytotoxic pathway was performed by pre-incubating NK cells for 2 h with 100 nM concanamycin A (CMA)(Sigma-Aldrich). Functional effects of ZB4 anti-Fas neutralizing mAb was also tested by pre-incubating target cells for 2 h before the assay.
7
Effector Cell Cytotoxicity Assay
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The effect of perforin or serine protease inhibitors on the cytotoxic activity of each effector cell population (CD8 + cells and CD8 -cells) was examined using concanamycin A (CMA, Sigma, St. Louis MO) or 3,4-dichloroisocoumarin (DCI, Sigma, St. Louis MO, USA), respectively. Each effector cell was separated from the gill and kidney of ginbuna carp as described above. Effector cells were treated with CMA at two concentrations (1 and 0.5 µM) for 2 h or DCI at two concentrations (40 and 20 µM) for 3 h at 22°C. The concentrations of these inhibitors were determined according to previous studies, and untreated effector cells were used as a control (Toda et al., 2011a (Toda et al., , 2011c)) . The inhibitortreated effector cells (9.0 × 10 4 cells) were then co-cultured with 300 I. multifiliis cells per well in 96-well plates in the presence of inhibitors for 2 h at 22°C. Dead parasites were then counted in each treated group at all inhibitor concentrations, and the percentage of dead cells was then calculated using the above formula, accounting for negative controls. The percentage of killing activity was calculated as described.
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