UniCAR T cells were cocultured with or without tumor spheroids in the presence or absence of CD98hc TM in a 96-well plate (E:T = 5:1) in complete RPMI medium with a total volume of 200 µL. After 48 h, cell culture plates were centrifuged for 5 min at 360× g, and cell-free supernatant was collected. IFN-γ concentrations were assessed by enzyme-linked immunosorbent assay (ELISA). Human IFN-Gamma ELISA Set and BD OptEIA Reagent Set B were obtained from BD Biosciences, Heidelberg, Germany. Additionally, intracellular staining of granzyme B was performed after 48 h as described previously [55 (link),61 (link),62 (link)]. Shortly, 100 μL of cocultured UniCAR T cells were transferred to a new 96-well plate. Extracellular staining, fixation, and permeabilization, as well as intracellular staining, were performed subsequently.
Human ifn gamma elisa set
Manufactured by BD
Sourced in Germany
The Human IFN-Gamma ELISA Set is a laboratory equipment used to measure the concentration of interferon-gamma (IFN-γ) in human samples. It is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed to accurately detect and quantify IFN-γ levels in various human biological samples, such as cell culture supernatants, serum, and plasma.
Automatically generated - may contain errors
Lab products found in correlation
Opteia reagent set b, by BD (3 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (2 mentions)
Human il 2 elisa set, by BD (2 mentions)
Human tnf elisa set, by BD (2 mentions)
Cell proliferation dye efluor 670, by Thermo Fisher Scientific (1 mentions)
Propidium iodide pbs solution, by Thermo Fisher Scientific (1 mentions)
3 protocols using human ifn gamma elisa set
1
Evaluating UniCAR T Cells' Function
2
Cytokine Release and Proliferation of UniCAR T Cells
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UniCAR T cells were cultured with or without tumor cells in the presence or absence of 50 nM TM in a 96-well plate (E:T = 5:1). Total volume was adjusted with RPMI complete media to 200 μl. After 48 h, cell culture plates were centrifuged for 5 min at 360xg and cell-free supernatant was collected. Concentration of TNF, IFN-γ, IL-2 and GM-CSF was determined by enzyme-linked immunosorbent assay (ELISA). Human TNF ELISA Set (#555212), Human IFN-Gamma ELISA Set (#555142), Human IL-2 ELISA Set (#555190), Human GM-CSF ELISA Set (#555126) as well as BD OptEIA Reagent Set B (#550534) were obtained from BD Biosciences. In addition, intracellular staining of perforin and granzyme B was performed after 48 h as described previously.49 –51 In order to monitor UniCAR T cell numbers in co-cultures, cells were labeled with Cell Proliferation Dye eFluor®670 (ThermoFisher Scientific, #65-0840-85) according to previously published protocols.49 ,52 After 24 h and 96 h of incubation, 20 μl of samples was transferred to a 96-well plate and mixed with 80 μl of 1 μg/ml propidium iodide/PBS solution (ThermoFisher Scientific, #P3566). Cell numbers were calculated using a MACSQuant Analyzer 10 (Miltenyi Biotec GmbH).
3
Cytokine Secretion and T Cell Activation in UniCAR-Mediated Tumor Targeting
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5 × 104 UniCAR T cells were incubated alone or in the presence of 1 × 104 tumor cells in a 96-well U-bottom plate. Recombinant Abs were added at a concentration of 5 nM. After 24 h, cell-free supernatant was harvested and analyzed for secreted cytokines by ELISA as previously described [45 (link)]. Human TNF ELISA Set, Human IFN-Gamma ELISA Set, Human IL-2 ELISA Set, and BD OptEIA Reagent Set B were purchased from BD Biosciences (Becton Dickinson GmbH, Heidelberg, Germany) and used according to the manufacturer’s instructions.
After 24 h of co-cultivation, UniCAR T cells were further examined with respect to their activation status. For this purpose, cells of one triplicate were pooled and stained with anti-human CD3-PE-Vio®770 or CD3-VioBlue and anti-human CD69-APC Abs (all Miltenyi Biotec GmbH) for 15 min at 4 °C. To allow exclusion of dead cells during analysis, counterstaining with 1 μg/mL propidium iode/PBS solution was performed. Flow cytometric data were acquired with the MACSQuant Analyzer 10 (Miltenyi Biotec GmbH).
After 24 h of co-cultivation, UniCAR T cells were further examined with respect to their activation status. For this purpose, cells of one triplicate were pooled and stained with anti-human CD3-PE-Vio®770 or CD3-VioBlue and anti-human CD69-APC Abs (all Miltenyi Biotec GmbH) for 15 min at 4 °C. To allow exclusion of dead cells during analysis, counterstaining with 1 μg/mL propidium iode/PBS solution was performed. Flow cytometric data were acquired with the MACSQuant Analyzer 10 (Miltenyi Biotec GmbH).
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