Cultured neural retina from 6-week-old animals was prepared as previously described (Chen et al., 2013b (link)). Eyes enucleated from mice were washed in a penicillin-streptomycin solution (50 unit/ml) (Sigma-Aldrich) and soaked in Hanks-balanced salt solution (Hyclone, Waltham, MA). Eye cups were flattened by slitting the peripheral side. Sterilized filter paper (5 × 5 mm) was placed on the neural retina side of the flattened-eye cup and the paper was carefully peeled away from the RPE/choroid. Each retina on the paper was put into a single well of a 12-well plate with 0.5 ml of DMEM containing 10% FCS and incubated for 16 h at 37°C. Retinas were further incubated with or without 30 μM atRAL for 6 h at 37°C after washing with 0.5 ml of DMEM containing 10% FCS twice. LDH assay was performed to determine amounts of retinal cell death (BioVision, Mountain View, CA). The percentage of cytotoxicity was calculated as [(a retina with atRAL – a retina without atRAL) / (lysis control – a retina without atRAL)] × 100.
Ldh assay
Manufactured by Abcam
Sourced in United States
The LDH assay is a colorimetric assay used to measure the activity of the enzyme Lactate Dehydrogenase (LDH) in biological samples. LDH is an enzyme found in various cell types and is involved in the conversion of lactate to pyruvate during cellular respiration. The assay quantifies the amount of LDH present, providing insights into cellular health and metabolism.
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Lab products found in correlation
Penicillin streptomycin solution, by Merck Group (1 mentions)
Procartaplex human immuno oncology checkpoint panel, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Ab102526, by Abcam (1 mentions)
Resazurin, by Thermo Fisher Scientific (1 mentions)
Resorufin, by Thermo Fisher Scientific (1 mentions)
Resazurin, by Merck Group (1 mentions)
Safire fluorescence plate reader, by Tecan (1 mentions)
10 protocols using ldh assay
1
Retinal Cell Death Assay
2
LDH Assay for Cell Death Quantification
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Cell death was determined using a lactate dehydrogenase (LDH) assay (Biovision, San Francisco, CA). Using an enzymatic coupling reaction, LDH was quantified in the supernatant and the tritonized cell lysate. LDH activity was determined by spectrophotometric reading at OD450. Percent cell death was calculated as concentration in supernatant/concentration in lysate × 100. Dilution at which 50% cell death would occur (lethal dose or LD50) was calculated assuming linear dose dependency (from studies in which testing of two doses concurrently was possible) with extrapolation of values out to a 1:10 dilution.
3
Blood Biomarker Analysis for Immunotherapy
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Blood collection and plasma preparation were performed as described previously 3 . Blood samples were taken before as well as between day 1 and day 4 after first administration of NCT. The exact day of blood collection had no statistical significant influence on the results 17 . Quantification of HMGB1 in the plasma samples was assessed by an enzyme‐linked immunosorbent assay (ELISA) kit (IBL Hamburg, Hamburg, Germany). Lactate dehydrogenase (LDH) levels were quantified using an LDH assay from Biovision (Mountain View, CA). Soluble forms of the checkpoint molecules CD27, CD28, CD80/B7‐1, CD137, CD152/CTLA‐4, CD223/LAG‐3, CD270/HVEM, CD272/BTLA, CD273/PD‐L2, CD274/PD‐L1, CD279/PD‐1, GITR, IDO, and TIM‐3 were measured using a ProcartaPlex® Human Immuno‐Oncology Checkpoint Panel (Affymetrix, Santa Clara, CA).
4
Adenovirus Vector Cytotoxicity and Cytokine Analysis
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The in vitro cytotoxicity was evaluated in ATDC5 cells by treating the cells with vehicle and adenovirus vector (Adv-RFP-Empty) by using Lactate dehydrogenase (LDH) assay as per the manufacturers’ instruction (Abcam Inc, Cambridge, MA, USA, ab102526). The serum samples of the mice injected with vehicle or adenovirus vector (Adv-RFP-Empty) were dilated 1:10 and 50 uL of serum samples may be evaluated for LDH assay (Abcam, Inc.). In order to evaluate the effect of the adenovirus vector on cytokine expression, RNA from the knee joint from the mice injected with vehicle or adenovirus vector (Adv-RFP-Empty) were subjected to RT-qPCR analysis (Taqman gene expression assay, Applied Biosystems, Waltham, MA, USA) for the expression of TNFα and IL6.
5
HMGB1 and LDH Serum Quantification
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Serum was collected 48 hours after treatment by saphenous bleed. The high mobility group box 1 protein (HMGB1) and lactate dehydrogenase (LDH) concentrations were determined using a mouse enzyme-linked immunosorbent assay kit (Antibodies online) and an LDH assay (Abcam), respectively, following manufacturers’ protocols.
6
MCF-7 Cells Viability Evaluation
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MCF-7 cells were seeded into 96-well plates (4800 cells/cm2) and incubated with different concentrations of ES medium, with CM and normal growth medium as control. Viability was evaluated using a resazurin assay on 4 consecutive days, where day 0 refers to the measurement prior to the start of treatment. Cells were incubated with normal growth medium supplemented with 0.07 µM of resazurin (Sigma-Aldrich) for two hours. Metabolic conversion of resazurin into the fluorescent resorufin was detected using a multiwell plate reader (excitation: 530 nm, emission: 590 nm, Varioskan, Thermo Fisher Scientific). Cytotoxicity was evaluated using the LDH Assay (abcam, Cambridge, UK) according to the manufacturers’ instructions.
7
Protein Antioxidant and Metabolic Assays
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Protein concentrations were normalized with a bovine serum albumin protein assay kit, and proteins were processed with a total antioxidant assay (BIOMEX, Seoul, Republic of Korea), TBARS assay (BIOMEX), glutathione colorimetric detection (ARBOR assay, Ann Arbor, MI, USA) and LDH assay (lactate dehydrogenase; LDH; Abcam), according to the manufacturer’s instructions. Experiments were performed in triplicate.
8
Assessing Cell Death Dynamics
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Cell death was assessed using an LDH Assay (Abcam). Cells were plated into a 96-well plate (20,000 cells per well) and the following day tissue media was exchanged to contain 200 mM of colbalt chloride and then incubated for 48 h. The assay was then performed as per the manufacturer’s protocol [24 ]. Three controls were used: (i) background control, providing a measure of absorbance from the culture media; (ii) a low control (no cell lysis); and (iii) a high control (10 µL of cell lysis solution (Triton x100) incubated in the tissue medium). Cell death = (sample absorbance – low control) / (high control – low control) x 100. Low control = normoxic environment. High control = Triton x100 lysis buffer for 10 min. Prior to the assay being conducted, the plate was centrifuged to remove any cell remnants from the tissue media and the media was transferred to a fresh 96-well plate.
9
Macrophage Viability Assay of Nanoparticles
10
Measuring Cellular LDH Secretion
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