MT-2, Jurkat and MT-1 cells were incubated with Clone D-IR700 and LAT-27-IR700 (0.01–1 μg/mL) at 37 °C for 1 h and irradiated with 10 J/cm2 of NIR light. After incubation for 1 h atRT, Cell titer-Glo (Promega) and LDH-Glo (Promega) were used for the cell viability and LDH assays, respectively. Each assay was performed according to the manufacturer’s protocol. Fluorescence was detected using a Glo-MAX plate reader (Promega).
Ldh glo
Manufactured by Promega
Sourced in United States
The LDH-Glo is a luminescent-based assay for the quantification of lactate dehydrogenase (LDH) activity. LDH is an enzyme commonly used as a marker for cell cytotoxicity and tissue damage. The LDH-Glo assay provides a sensitive and quantitative measurement of LDH levels in cell culture samples.
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Lab products found in correlation
Celltiter glo, by Promega (2 mentions)
Glomax plate reader, by Promega (1 mentions)
Pd 1 pd l1 blockade bioassay, by Promega (1 mentions)
Clariostar, by BMG Labtech (1 mentions)
Veritas, by Promega (1 mentions)
Human serum, by R&D Systems (1 mentions)
Recombinant human il 15, by R&D Systems (1 mentions)
Penicillin streptomycin, by R&D Systems (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Vybrant mtt, by Thermo Fisher Scientific (1 mentions)
Biotek synergy ht plate reader, by Agilent Technologies (1 mentions)
7 protocols using ldh glo
1
Photodynamic Cytotoxicity Assay
2
Cell Viability Assay for DDCs
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Cell viability was measured using CellTiter-Glo or LDH-Glo (G7570, J2380, Promega). Briefly, CD22 positive or negative cells were plated into 96-wells, allowing attachment and growth for 24 hr, then triplicate wells were treated with DDCs, naked antibodies, free drugs, or DDCs plus competitor antibodies. Three to five days later, when untreated control wells were 70 to 90% confluent, reagent was added to the plates according to the supplier’s instructions. Wells treated identically but wells without cells were used to subtract background. Fluorescence (ex: 570 nm, Em: 585 nm) was measured using a CLARIOstar microplate reader (BMG Labtech) and data analyzed using GraphPad Prism 8.0.1 software. Significance was tested using one-way ANOVA, followed by the Tukey’s multiple post hoc test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 between the indicated groups. PD-L1 neutralization was done in a PD-1/PD-L1 Blockade Bioassay (J1250, Promega).
3
Transepithelial Electrical Resistance and Cell Viability
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TEER was measured with a volt-ohmmeter and electrode probe (EVOM and STX2; World Precision Instruments, Sarasota, FL, USA). Details on obtaining reproducible resistance readings are available at: https://medicine.umich.edu/sites/default/files/content/downloads/Measuring_Transepithelial_Electrical_Resistance.pdf .
Cell death was measured on cultures at least several weeks after last OS feeding using a cytotoxicity assay (LDH-Glo, #J2380; Promega, Madison, WI, USA) according to manufacturer's instructions. Supernatant (24-hour incubation) was diluted 1:100, and 50 μL of the dilution was measured on a luminometer (Veritas; Promega), subtracting all values from the signal derived from fresh media.
Cell death was measured on cultures at least several weeks after last OS feeding using a cytotoxicity assay (LDH-Glo, #J2380; Promega, Madison, WI, USA) according to manufacturer's instructions. Supernatant (24-hour incubation) was diluted 1:100, and 50 μL of the dilution was measured on a luminometer (Veritas; Promega), subtracting all values from the signal derived from fresh media.
4
LDH-Based Cytotoxicity Assay Protocol
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LDH-GLO (J2381; Promega) cytotoxicity assay was performed by preserving culture medium from post-treatment timepoints of 4 h and 72 h, then running the assay on the same day according to manufacturer specifications to measure lactose dehydrogenase (LDH) enzymatic activity. Well contents for both assays were transferred to a Costar White Polystyrene 96 well Assay Plate and analyzed with a Veritas Microplate Luminometer.
5
Cytotoxicity and Degranulation Assays for Immune Cells
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For degranulation assays, anti-CD107a mAb was added to the co-cultures of tumor cells and effector cells. After 1 h of incubation, GolgiStop™ (BD) was added and incubated for four additional hours at 37°C. For in vitro cytotoxicity studies, FACS-sorted CD8+ T cells (NKp30+ or NKp30− bearing or not HER2TCR or HER2CAR) from the same donor were co-cultured with the indicated cell lines for 24 h at 37°C. Target cell killing was determined by using the LDH-Glo™ (Promega) assay kit. Target cells were lysed for maximum release determination. % specific lysis = 100× ((LDH release)-(spontaneous LDH release))/((maximum LDH release)-(spontaneous LDH release)). For functional assays, cells were cultured in RPMI 1640 medium (Sigma-Aldrich) with 10% human serum (PAA), 1% Penicillin/Streptomycin and recombinant human IL-15 (R&D Systems), as above described.
For determination of IFN-γ production, supernatants from effector and target cell co-cultures were collected and cytokine concentration was quantified using ELISA MAX™ Deluxe Set for Human IFN-γ (Biolegend), according to the manufacturer's instructions.
For determination of IFN-γ production, supernatants from effector and target cell co-cultures were collected and cytokine concentration was quantified using ELISA MAX™ Deluxe Set for Human IFN-γ (Biolegend), according to the manufacturer's instructions.
6
Cytotoxicity and Viability Assay in H9C2 Cells
7
LDH-Glo Cytotoxicity Assay for Hydrogels
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Cytotoxicity was quantified using the LDH-Glo™ (Promega, J2380) as described by the manufacturer's instructions. Briefly, samples were prepared by combining 4 μL of medium per hydrogel and diluting it in 96 μL LDH (lactate dehydrogenase) storage buffer (200 mM Tris-HCl (Roche, 10812846001), 10% glycerol (Promega, H5433), 1% bovine serum albumin (BSA; Sigma-Aldrich, A4503); filter sterilized and stored at 4°C). This dilution was calculated to fall within the linear regime of the assay (data not shown). Samples were used immediately or stored at -80°C until use. Prior to running the assay, all reagents were equilibrated to room temperature. Duplicate wells were prepared for each hydrogel by adding 50 μL of sample and 50 μL prepared LDH Detection Reagent per well in 96-well opaque, white-walled plates.
Samples were protected from light and incubated at room temperature for 60 minutes. After 60 minutes, luminescence was read immediately using a plate reader with a 1 second integration time (BioTek Synergy HT Plate Reader and Gen5 Software, BioTek Instruments, Inc.). Relative luminescence for each sample was calculated by subtracting the luminescence from the sample blank (cell medium) from each hydrogel sample.
Samples were protected from light and incubated at room temperature for 60 minutes. After 60 minutes, luminescence was read immediately using a plate reader with a 1 second integration time (BioTek Synergy HT Plate Reader and Gen5 Software, BioTek Instruments, Inc.). Relative luminescence for each sample was calculated by subtracting the luminescence from the sample blank (cell medium) from each hydrogel sample.
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