ADCC was determined using DELFIA EuTDA cytotoxicity assay as we have previously described [45 (link)]. 1 × 106 target cells were labeled with BATDA labeling ligand for 30 minutes. Tumor target cells were washed and incubated with 10 μg/ml obinutuzumab, rituximab or polyclonal IgG at 37°C and incubated with expanded NK effector cells, at 10:1 and 5:1 E: T target ratio for 4 hours at 37°C. Plates were centrifuged and the supernatant was collected into a flat-bottom plate and Europium solution was added and incubated for 15 minutes at room temperature. Fluorescence was measured with a time-resolved fluorometer (Perkin Elmer). The percentage of cytotoxicity was calculated using following equation: % specific release = (E-S/(M-S) × 100) E = experimental release, S = spontaneous release (target cells without effector cells), M = maximum release (lysed target cells).
Time resolved fluorometer
Manufactured by PerkinElmer
Sourced in United States
The Time-resolved fluorometer is an instrument used to measure the fluorescence of samples over time. It detects and analyzes the emission of light from fluorescent molecules after they have been excited by a light source. The instrument provides precise and accurate measurements of the fluorescence intensity and decay characteristics of the sample.
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Lab products found in correlation
4 protocols using time resolved fluorometer
1
ADCC Assay Protocol with Obinutuzumab, Rituximab, and IgG
2
Tumor Cytotoxicity Assay with NK Cells
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Since there were few CD56bright cells, further studies were
performed on PB and CB CD56dim subsets only. Tumor cytotoxicity
was compared between PB and CB NK CD56dim against tumor targets
as determined by europium release assay (Perkin Elmer, Waltham, MA, USA), as
we have described.40 (link) Tumor cytotoxicity was measured against an anaplastic large cell
lymphoma cell line (ALCL), Karpas-299 (DSMZ, Germany), and a diffuse large
B-cell lymphoma cell line (DLBCL), Toledo (ATCC , Manhass, VA).
BATDA (acetyoxymethyl ester-enhancing ligand)-labeled tumor targets were
re-suspended at a final concentration of 1 × 105 cells/ml. CB and
PB NK CD56dim effector cells were added to 5 × 104labeled tumor targets at varying effector:target (E:T) ratios (5:1, 10:1 and
20:1) for 2 h at 37°C, 5% CO2. After incubation, cytolytic
activity was evaluated using a time-resolved fluorometer (Perkin Elmer,
Waltham, MA, USA). Percentage of specific release was calculated as follows:
Spontaneous release was determined by incubating tumor targets in medium
alone and the maximum release was determined by incubating tumor targets in
medium + lysis buffer. Cytotoxicity was expressed as percent specific
release. All tests were run in triplicate.
performed on PB and CB CD56dim subsets only. Tumor cytotoxicity
was compared between PB and CB NK CD56dim against tumor targets
as determined by europium release assay (Perkin Elmer, Waltham, MA, USA), as
we have described.40 (link) Tumor cytotoxicity was measured against an anaplastic large cell
lymphoma cell line (ALCL), Karpas-299 (DSMZ, Germany), and a diffuse large
B-cell lymphoma cell line (DLBCL), Toledo (
BATDA (acetyoxymethyl ester-enhancing ligand)-labeled tumor targets were
re-suspended at a final concentration of 1 × 105 cells/ml. CB and
PB NK CD56dim effector cells were added to 5 × 104labeled tumor targets at varying effector:target (E:T) ratios (5:1, 10:1 and
20:1) for 2 h at 37°C, 5% CO2. After incubation, cytolytic
activity was evaluated using a time-resolved fluorometer (Perkin Elmer,
Waltham, MA, USA). Percentage of specific release was calculated as follows:
Spontaneous release was determined by incubating tumor targets in medium
alone and the maximum release was determined by incubating tumor targets in
medium + lysis buffer. Cytotoxicity was expressed as percent specific
release. All tests were run in triplicate.
3
Quantitative Fluorometric ELISA for VEGF and NGAL
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TRIFMA was performed as described earlier [101 (link),102 (link)]. In brief, microtiter 96-well plates were coated overnight at 4 °C with primary antibodies dissolved in PBS. They were specific for either NGAL (1 µg/mL) or VEGF (2 µg/mL). All wells were then incubated with blocking buffer (1% Tween-20 in PBS) for 2 h.
The samples were diluted 1:2 in assay buffer to a total of 100 µL and added to the plates. The assay buffer used was PBS supplemented with 0.05% Tween-20. In addition, recombinant standards (VEGF and NGAL) were added to the plate (100 µL per well). Negative control samples, containing 100 µL of assay buffer without added supernatant, were also added to the plates. All samples were added to the plates in duplicate to increase the precision of the results.
The Fluorescence was measured using a time-resolved fluorometer (PerkinElmer, Waltham, MA, USA). The concentrations of VEGF and NGAL were determined using a 4-parameter standard curve fit implemented in WorkOut 2.5 data analysis software (PerkinElmer, Waltham, MA, USA).
The samples were diluted 1:2 in assay buffer to a total of 100 µL and added to the plates. The assay buffer used was PBS supplemented with 0.05% Tween-20. In addition, recombinant standards (VEGF and NGAL) were added to the plate (100 µL per well). Negative control samples, containing 100 µL of assay buffer without added supernatant, were also added to the plates. All samples were added to the plates in duplicate to increase the precision of the results.
The Fluorescence was measured using a time-resolved fluorometer (PerkinElmer, Waltham, MA, USA). The concentrations of VEGF and NGAL were determined using a 4-parameter standard curve fit implemented in WorkOut 2.5 data analysis software (PerkinElmer, Waltham, MA, USA).
4
Cytolytic Activity Assay for Tumor Cells
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Tumor cytolytic activity was determined by europium release assay as determined with a standard kit (Perkin Elmer, Wellesley, MA, USA), as we have described previously [21] . Tumor cytotoxicity was measured against a BL cell line, Ramos (ATCC), and a DLBCL cell line, SUDHL-6 (DSMZ). An NK-sensitive human erythroleukemia cell line, K562 (ATCC), was used as a positive control. BATDA (acetyoxymethyl ester-enhancing ligand)-labeled tumor targets were resuspended at a final concentration of 1 Â 10 5 cells/mL. CB MNCs EvE with MODK562 cells or WTK562 were added to 5 Â 10 4 labeled tumor targets at a 20:1 E:T ratio and incubated for 2 hours at 37 C and 5% CO 2 . After incubation, cytolytic activity was evaluated using a timeresolved fluorometer (Perkin Elmer). The percentage of specific release was calculated as follows: %specific release
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