For generating Hepa 1-6–immunized splenocytes, 8-week-old C57BL/6 mice were immunized with subcutaneous inoculation of 104 Hepa 1-6 cells once a week for 3 cycles. After immunizing, they were killed humanely, and a routine surgical procedure was used to aseptically remove the spleen. Then, single-cell suspensions were prepared by mechanically passing spleen tissue through mesh screens. The erythrocytes were lysed, and the cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin. For the cytotoxicity assay of Hepa 1-6–immunized splenocytes, 104 target cells were seeded in 96-well plates. After 12 hours, allowing for adhesion, the target cells were cocultured with splenocytes for 5 hours; the stands for effector cell to target cell (E/T) ratios were respectively 20, 40, and 80. The cytotoxic activities of splenocytes in the presence of target cells were analyzed with an lactate dehydrogenase (LDH) cytotoxicity assay kit (BioVision).
Lactate dehydrogenase ldh cytotoxicity assay kit
Manufactured by Abcam
Sourced in United States
The Lactate dehydrogenase (LDH) cytotoxicity assay kit is a colorimetric assay that quantitatively measures the release of lactate dehydrogenase, an enzyme present in the cytoplasm of cells. The assay is used to determine cell cytotoxicity or cell death.
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3 protocols using lactate dehydrogenase ldh cytotoxicity assay kit
1
Generating Hepa 1-6-Immunized Splenocytes
2
LDH Cytotoxicity Assay for Membrane Damage
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The Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit (BioVision, Mountain View, CA, USA) was used for quantification of plasma membrane damage. MLO-Y4 cells were seeded in a 96-well plate overnight at 37°C. After treatment with Dex for 24hrs, the plates were centrifuged at 250g for 10mins and 100μl of supernatant was transferred into corresponding wells of a clear 96-well plate. To the supernatant, 100μl of reaction mixture was added to each well and incubated for 30mins in the dark. The absorbance at 490nm was then measured using a spectrophotometer microplate reader BioRAD spectrophotometer (Model 680). As positive control, the maximum release of LDH was determined by incubation of the cells in 1% Triton X-100 for 1hr in culture medium.
3
Investigating Gastric Adenocarcinoma Cell Viability
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The AGS cell line (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) was originally cultured from stomach adenocarcinoma cells obtained prior to any anti-cancer treatment (Barranco et al., 1983) . Sodium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). The Quick Cell Proliferation Assay and Annexin V-PE (phycoerythrin) Apoptosis Detection kits were purchased from Medical & Biological Laboratories (Aichi, Japan). The lactate dehydrogenase (LDH)-cytotoxicity assay kit was purchased from Biovision (Milpitas, CA, USA) and the ApoAlert DNA Fragmentation Assay kit was purchased from BD Biosciences (Franklin Lakes, CA, USA). Caspase-3, -6, -8, and -9 colorimetric protease assays were purchased from Beyotime (Shanghai, China). The DNA Fragmentation Assay kit was purchased from BDBiosciences. Ham's F12 medium and fetal bovine serum were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA).
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