Ldh cytotoxicity colorimetric assay kit

Cytotoxicity was quantitatively evaluated by examining the release of LDH in the medium. As high control, cells (passage numbers 13~14, 27~29) were pretreated for 1 h with 1% (v/v) Triton X-100 in the culture medium. All procedures were carried out as per the supplier’s instructions (LDH cytotoxicity Colorimetric Assay Kit, BioVision). In brief, after treatment, 100 µL of culture medium from each well was transferred to a new 96-well plate, 100 µL of reaction mixture was then added and incubated at room temperature for 30 min. The release of LDH was measured by noting the optical density (OD) at 490 nm using a microplate ELISA reader. LDH release into the medium was calculated using the following equation:

Cell Counting Kit-8 (CCK-8, Sigma-Aldrich) was used to detect the viability of astrocytes and neurons. An LDH-Cytotoxicity Colorimetric Assay Kit (Biovision) was used to analyze lactate dehydrogenase (LDH) release in the medium.

Induced egress was performed as described previously (15 (link)). Briefly, parasites grown in HFFs in an 8-well chamber slide for 30 h were treated with 1% dimethyl sulfoxide, 2 μM A23187, or 200 μM zaprinast in assay buffer (Hanks' buffered salt solution containing 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES) and incubated for 2 min in a 37°C water bath. Egress was stopped by addition of 2× fixative (8% formaldehyde in 1× PBS). Immunofluorescence was performed with rabbit anti-SAG1 to identify parasites and mouse anti-GRA7 (a generous gift from Peter Bradley) to identify the parasitophorous vacuole membrane. At least 10 fields of view (400× total magnification) per condition were enumerated as occupied or unoccupied.
LDH egress assays were performed as previously described (20 (link)). Briefly, parasites were grown in HFF monolayers in 96-well plates for 30 h. Wells were washed with Ringer’s buffer and then treated with 100 μM zaprinast diluted in Ringer’s buffer. Plates were incubated at 37ºC for 20 min before removal and placement on ice. Fifty microliters of the supernatant were removed and centrifuged in a separate round-bottom plate. Thirty microliters of supernatant was subsequently removed and release of lactate dehydrogenase was determined using an LDH cytotoxicity colorimetric assay kit (BioVision).

A LDH-Cytotoxicity Colorimetric Assay Kit (BioVision) was used to evaluate the cytotoxicity of MnO₂ NSs and MBP-Ce6 NSs. Human normal prostatic myofibroblast (WPMY-1) cells were cultured in DMEM (Dulbecco's modified Eagle's medium, KeyGen, BioTech) with 10% fetal bovine serum (FBS, Gibco) medium at 37°C under 5% CO₂. WPYM-1 cells were added in 96-well plates with the concentration of 10⁴ cell per well. After being cultured for 24 h, the supernatant medium was removed and MnO₂ NS and MBP NS dispersions (200 μL, suspended in DMEM (without FBS)) containing different concentrations of MnO₂ (0, 20, 40, 80, 160, and 320 μg/mL) were added into the 96-well plate. Cells cultured without any nanosheets were set as low control, and those cultured with 1% Triton X-100 were used as high control. After 24 h, 100 μL of supernatant in each well was transferred into another 96-well plate, and 100 μL reaction solution was added into the corresponding well. After 30 min, a microplate reader (PowerWave XS2, BioTek) was used to measure the optical density at 495 nm (OD₄₉₅). The viability of cells was calculated according to the following formula
$\begin{array}{c}\text{Cell\hspace{0.17em}viability}=100\%-\left(A_{\text{T}}–A_{\text{L}}\right)/\left(A_{\text{H}}–A_{\text{L}}\right)\times 100\%\end{array}$ where A_T represents the OD₄₉₅ of test sample, A_L represents the OD₄₉₅ of low control, and A_H represents the OD₄₉₅ of high control.

LDH assay was carried out using a LDH-cytotoxicity colorimetric assay kit (Bio-Vision Inc., Milpitas, CA). In brief, 1 × 10⁴cells/well were seeded in 96-well plates and exposed to different concentrations of NPs for 24 h. After the exposure period had elapsed, each 96-well plate was centrifuged at 2300 g for 5 min to settle the NPs present in the solution. Then 100 μl of the supernatant was transferred to new 96-well plate that already contained 100 μl of the reaction mixture from the BioVision kit and incubated for 30 min at room temperature. At the end of incubation time, absorbance of the solution was measured at 340 nm using the microplate reader (Synergy-HT, BioTek, Winooski, VT). The LDH levels in the culture medium versus those in the cells were quantified and compared with the control values according to the manufacturer of the kit’s instructions.

LDH levels in the cultured medium were determined by LDH-Cytotoxicity Colorimetric Assay Kit (Biovision, Milpitas, CA). The absorbance was measured at 490 nm using a microplate reader (BioTek, Winooski, Vermont) according to the manufacturer’s instructions.