Caspase3/9 activity (Beyotime, China), LDH release (Beyotime, China) and TUNEL late-apoptotic (Roche Applied Bio Sciences, USA) analyses were performed using commercial kits as previously described [28] (link).
Ldh release
Manufactured by Beyotime
Sourced in China
The LDH release assay is a laboratory tool used to quantify the amount of lactate dehydrogenase (LDH) released from cells. LDH is an enzyme found in the cytoplasm of cells, and its release is an indicator of cell membrane damage or cell death. The assay provides a measurement of LDH activity in cell culture media or other biological samples, allowing researchers to assess cellular cytotoxicity or viability.
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Lab products found in correlation
4 protocols using ldh release
1
Apoptosis Assays in Cell Lines
2
NLRP3 Inflammasome Activation in BMDMs and THP-1 Cells
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BMDMs were isolated from bone marrow and cultured for 6-7 days in DMEM supplemented with 10% FBS, 1% antibiotics and 20 ng/mL M-CSF.
THP-1 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% antibiotics.
To activate the NLRP3 inflammasome, BMDMs (5×105 cells/mL) or THP-1 cells (1×106 cells/mL) were planted in 12-well plates, THP-1 cells were treated with PMA (100nM) for one night, and the overnight culture media was replaced with Opti-MEM the following morning. After three hours of priming with 50 ng/mL LPS, BMDMs were stimulated with apilimod or treated with other agents (MCC950, CY09, MnTBAP, Baf-A1, or CA-074-Me) according to the experimental needs for 30 min before the stimulation of apilimod. THP-1 cells were stimulated with apilimod after LPS-priming. Using Lipofectamine 2000, cells were transfected with poly(A/T) (0.5 g/mL) for 2 h. Immunoblotting was used to evaluate supernatants and cell extracts that had been precipitated. Using an LDH Cytotoxicity Assay Kit, LDH release was quantified (Beyotime).
THP-1 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% antibiotics.
To activate the NLRP3 inflammasome, BMDMs (5×105 cells/mL) or THP-1 cells (1×106 cells/mL) were planted in 12-well plates, THP-1 cells were treated with PMA (100nM) for one night, and the overnight culture media was replaced with Opti-MEM the following morning. After three hours of priming with 50 ng/mL LPS, BMDMs were stimulated with apilimod or treated with other agents (MCC950, CY09, MnTBAP, Baf-A1, or CA-074-Me) according to the experimental needs for 30 min before the stimulation of apilimod. THP-1 cells were stimulated with apilimod after LPS-priming. Using Lipofectamine 2000, cells were transfected with poly(A/T) (0.5 g/mL) for 2 h. Immunoblotting was used to evaluate supernatants and cell extracts that had been precipitated. Using an LDH Cytotoxicity Assay Kit, LDH release was quantified (Beyotime).
3
SPIO Nanoclusters Cytotoxicity Evaluation
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The three types of cells were seeded in 96-well plates at a density approximate 1 × 104 cells/well respectively and incubated with various concentrations of SPIO nanoclusters for 24 h. LDH release was measured in cell-free medium following the manufacturer’s instructions (Beyotime, China).
4
Cytotoxicity Assay of Stem Cell-Derived NK Cells
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LDH release method (LDH Cytotoxicity Assay) was used to detect the direct cytotoxicity of derived SEs-NK cells to target cells. SEs-NK cells were used as the effector cells, and K562 cells were used as the target cells in the cytotoxicity assay. The effector-target ratios (E: T) were 20:1, 50:1, 80:1, and 100:1. Cytotoxicity was determined by LDH release from cells according to the manufacturer's instructions (Beyotime). The resultant LDH release was analyzed using an ELISA plate reader (Thermo Scienti c). The cytotoxicity was calculated by the following formula: Statistical analysis Data were analyzed using GraphPad Prism 8 (GraphPad Software). Student's t-tests and one-way analysis of variance (ANOVA) were used to analyze between two groups or among multiple groups, respectively. All experiments were repeated three times and data were shown as mean±standard error of mean (SEM). Differences were considered statistically signi cant when P < 0.05 (* P0.05, ** P0.01, *** P0.001).
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