Raji cells were seeded onto a 96-well white assay plates with clear bottom at 10,000 cells/well in 1% BSA in phenol free RPMI medium. The test antibodies (anti-CD20 x anti-C1q bsAb, anti-CD20 bivalent hIgG1 antibody, Rituximab and anti-C1q bivalent hIgG1 antibody) were serially diluted 1:8 from 100 nM to 0.004 nM and added to the cells, along with human serum at a final concentration of 5%. Cells were incubated for 1 h at 37 °C and in 5% CO2 and cytotoxicity was measured using the CytoTox-Glo reagent (Promega) according to the manufacturer’s instructions. Results are plotted as mean ± standard deviation.
Cytotox glo reagent
Manufactured by Promega
Sourced in United States
The CytoTox-Glo reagent is a luminescent assay that measures the number of dead cells in a cell population. It detects a protease marker released from dead cells, providing a quantitative measure of cytotoxicity.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 binding, by BD (2 mentions)
White 96 well plate, by Corning (2 mentions)
Propidium iodide, by BD (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Cytexpert software, by Beckman Coulter (2 mentions)
Adcc reporter bioassay kit, by Promega (1 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
Clariostar plus, by BMG Labtech (1 mentions)
11 protocols using cytotox glo reagent
1
Antibody-Mediated Complement-Dependent Cytotoxicity
2
Complement-Dependent Cytotoxicity Assay
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Example 7
Cynomolgus cross-reactive clones were tested for the ability to induce complement-dependent cytotoxicity (CDC). MOLP-8 cells were plated at a density of 10,000 cells per well in a black 96-well flat-bottom tissue culture plate in 50 μL of complete media (RPMI supplemented with 10% fetal bovine serum). 50 μL of 2× anti-CD38 antibody, control IgG antibody, or media alone was added to each well and left to incubate at room temperature for 10 min. Varying amounts (2-15 μL) of purified rabbit complement (cat #CL 3441 Cedarlane Laboratories, Canada), depending upon the cell line was added to each well except control wells. After one hour incubation at 37° C., plates were brought to room temperature, 100 μL of cell titer CytoTox Glo™ reagent (Promega G7571/G7573) was added per well, the plate shaken for 5 to 7 min and luminescence read on an EnVision® (Perkin Elmer) luminescence plate reader. Conditions tested: cells alone; cells+complement; cells+IgG control+complement; cells+antibody+complement. % CDC was calculated using the following equation:
100−(RLUT/RLUC)×100),
where RLUT is the relative luminescence units of the test sample and RLUC is the relative luminescence units of the sample with complement alone. Statistical analysis was performed using PRISM software. EC50 values, as determined from plots of % CDC versus antibody concentration, are shown in Table 1.
3
Complement-Dependent Cytotoxicity Assay for Anti-CD38 Antibodies
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Example 7
Cynomolgus cross-reactive clones were tested for the ability to induce complement-dependent cytotoxicity (CDC). MOLP-8 cells were plated at a density of 10,000 cells per well in a black 96-well flat-bottom tissue culture plate in 50 μL of complete media (RPMI supplemented with 10% fetal bovine serum). 50 μL of 2× anti-CD38 antibody, control IgG antibody, or media alone was added to each well and left to incubate at room temperature for 10 min. Varying amounts (2-15 μL) of purified rabbit complement (cat #CL 3441 Cedarlane Laboratories, Canada), depending upon the cell line was added to each well except control wells. After one hour incubation at 37° C., plates were brought to room temperature, 100 μL of cell titer CytoTox Glo™ reagent (Promega G7571/G7573) was added per well, the plate shaken for 5 to 7 min and luminescence read on an EnVision® (Perkin Elmer) luminescence plate reader. Conditions tested: cells alone; cells+complement; cells+IgG control+complement; cells+antibody+complement. % CDC was calculated using the following equation:
100−(RLUT/RLUC)×100),
where RLUT is the relative luminescence units of the test sample and RLUC is the relative luminescence units of the sample with complement alone. Statistical analysis was performed using PRISM software. EC50 values, as determined from plots of % CDC versus antibody concentration, are shown in Table 1.
4
Complement-Dependent Cytotoxicity Assay
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Example 7
Cynomolgus cross-reactive clones were tested for the ability to induce complement-dependent cytotoxicity (CDC). MOLP-8 cells were plated at a density of 10,000 cells per well in a black 96-well flat-bottom tissue culture plate in 50 μL of complete media (RPMI supplemetned with 10% fetal bovine serum). 50 μL of 2× anti-CD38 antibody, control IgG antibody, or media alone was added to each well and left to incubate at room temperature for 10 min. Varying amounts (2-15 μL) of purified rabbit complement (cat #CL 3441 Cedarlane Laboratories, Canada), depending upon the cell line was added to each well except control wells. After one hour incubation at 37° C., plates were brought to room temperature, 100 μL of cell titer CytoTox Glo™ reagent (Promega G7571/G7573) was added per well, the plate shaken for 5 to 7 min and luminescence read on an EnVision® (Perkin Elmer) luminescence plate reader. Conditions tested: cells alone; cells+complement; cells+IgG control+complement; cells+antibody+complement. % CDC was calculated using the following equation:
100−(RLUT/RLUC)×100),
where RLUT is the relative luminescence units of the test sample and RLUC is the relative luminescence units of the sample with complement alone. Statistical analysis was performed using PRISM software. EC50 values, as determined from plots of % CDC versus antibody concentration, are shown in Table 1.
5
ADCC and CDC Activity Assay
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ADCC activity was determined using ADCC reporter bioassay kit (Promega) following the protocol provided by the supplier. For CDC assays, target cells were prepared at a density of 1.0×105 cells/mL and plated 100 µL to each well in the 96-well plate. Serially diluted antibodies were then added to the wells and 25 µL of twofold diluted human complement serum (Quidel) was added to the well followed by incubation for 5 hours at 37°C. Rituximab was used as a positive control. The detection of luminescence was performed by a plate reader (PHERAstar) following the addition of CytoTox-Glo reagent (Promega).
6
Anti-GITR x Anti-C1q Antibody CDC Assay
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The ability of the anti-GITR x anti-C1q antibody to mediate CDC in the presence of NHS was evaluated in engineered Jurkat/hGITR/hCD20 target cells. NHS was added at a final serum concentration of 5% to target cells seeded at 5,000 cells/well in triplicate rows of a 96-well white plate. Serial dilutions of anti-GITR x anti-C1q antibodies ranging from 0.5 pM to 500 nM were then added to target cells. Cells were incubated for 3.5 h at 37 °C and 5% CO2 and cytotoxicity was measured using the CytoTox-Glo reagent (Promega) according to the manufacturer’s instructions. Results are plotted as mean ± standard deviation.
7
Complement-Dependent Cytotoxicity Assay
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Example 7
Cynomolgus cross-reactive clones were tested for the ability to induce complement-dependent cytotoxicity (CDC). MOLP-8 cells were plated at a density of 10,000 cells per well in a black 96-well flat-bottom tissue culture plate in 50 μL of complete media (RPMI supplemented with 10% fetal bovine serum). 50 μL of 2× anti-CD38 antibody, control IgG antibody, or media alone was added to each well and left to incubate at room temperature for 10 min. Varying amounts (2-15 μL) of purified rabbit complement (cat #CL 3441 Cedarlane Laboratories, Canada), depending upon the cell line was added to each well except control wells. After one hour incubation at 37° C., plates were brought to room temperature, 100 μL of cell titer CytoTox Glo™ reagent (Promega G7571/G7573) was added per well, the plate shaken for 5 to 7 min and luminescence read on an EnVision® (Perkin Elmer) luminescence plate reader. Conditions tested: cells alone; cells+complement; cells+IgG control+complement; cells+antibody+complement. % CDC was calculated using the following equation:
100−(RLUT/RLUC)×100),
where RLUT is the relative luminescence units of the test sample and RLUC is the relative luminescence units of the sample with complement alone. Statistical analysis was performed using PRISM software. EC50 values, as determined from plots of % CDC versus antibody concentration, are shown in Table 1.
8
MTT Viability, Apoptosis, and Cytotoxicity Assays
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[3–5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt] MTT cell viability assays were performed in 96-well dishes using Cell Titer 96 Aqueous One solution reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Data are expressed as a percentage of mock-infected cells analyzed in parallel.
Alternatively, cells cultured in 24-well plates (2 cm diameter) were treated as indicated in each figure legend (concentration of drug and time). Media and trypsinized adherent cells were centrifuged and analyzed for annexin V binding (BD Bioscience, San Jose, CA, USA) and propidium iodide (BD Bioscience) staining as described by the manufacturer. Cells were analyzed by flow cytometry using the CytoFLEX (Beckman Coulter, Brea, CA, USA) and 10,000 independent events were analyzed using CytExpert software (Beckman Coulter).
Cytotoxicity assays were performed in 96-well white plates (Corning, Corning, NY, USA) using CytoTox-Glo reagent (Promega) according to the manufacturer’s instructions. Data are expressed as a fold change of mock-infected cells analyzed in parallel. Functional caspase assays were performed in 96-well white plates (Corning) using Caspase-Glo 9, 8, or 3/7 assays (Promega) according to the manufacturer’s instructions. Data are expressed as a fold change of mock-infected cells analyzed in parallel.
Alternatively, cells cultured in 24-well plates (2 cm diameter) were treated as indicated in each figure legend (concentration of drug and time). Media and trypsinized adherent cells were centrifuged and analyzed for annexin V binding (BD Bioscience, San Jose, CA, USA) and propidium iodide (BD Bioscience) staining as described by the manufacturer. Cells were analyzed by flow cytometry using the CytoFLEX (Beckman Coulter, Brea, CA, USA) and 10,000 independent events were analyzed using CytExpert software (Beckman Coulter).
Cytotoxicity assays were performed in 96-well white plates (Corning, Corning, NY, USA) using CytoTox-Glo reagent (Promega) according to the manufacturer’s instructions. Data are expressed as a fold change of mock-infected cells analyzed in parallel. Functional caspase assays were performed in 96-well white plates (Corning) using Caspase-Glo 9, 8, or 3/7 assays (Promega) according to the manufacturer’s instructions. Data are expressed as a fold change of mock-infected cells analyzed in parallel.
9
ADCC Assay with KVA Antibodies
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A dose-titration was performed to test KVA antibodies (100 pg/mL - 10 mg/mL) in the ADCC assay with Effector cells (PBMCs from healthy donors treated with 200 U/mL of IL-2 O/N) and Target cells (hVISTA-Raji cells) with a [12:1] ratio. Cell death was detected using CytoToxGlo™ reagent (Promega) after a 4-hour incubation. Relative luminescent units (RLU) were measured using a ClarioStar Plus plate reader (BMG Labtech). A fold increase of dead cells over untreated negative control was plotted.
10
Annexin V/PI Flow Cytometry Protocol
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Cells cultured in 24-well plates or 48-well plates were treated as indicated in each figure legend. Media and trypsinized adherent cells were centrifuged and analyzed for annexin V binding (BD Bioscience, San Jose, CA, USA) and propidium iodide (BD Bioscience) staining as described by the manufacturer. Cells were analyzed by flow cytometry using the CytoFLEX (Beckman Coulter, Brea, CA, USA) and 10,000 independent events were analyzed using CytExpert software (Beckman Coulter).
Alternatively, cytotoxicity assays were performed in 96-well white plates (Corning, Corning, NY, USA) using CytoTox-Glo reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Data are expressed as a fold change over mock-infected cells analyzed in parallel. Functional caspase assays were performed in 96-well white plates (Corning) using Caspase-Glo 9, 8, or 3/7 assays (Promega) according to the manufacturer’s instructions. Data are expressed as a fold change over mock-infected cells analyzed in parallel.
Alternatively, cytotoxicity assays were performed in 96-well white plates (Corning, Corning, NY, USA) using CytoTox-Glo reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Data are expressed as a fold change over mock-infected cells analyzed in parallel. Functional caspase assays were performed in 96-well white plates (Corning) using Caspase-Glo 9, 8, or 3/7 assays (Promega) according to the manufacturer’s instructions. Data are expressed as a fold change over mock-infected cells analyzed in parallel.
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