Automated assay to determine hydrogen peroxide levels in THP-1 cells on OT-2

Dr. Vasudha SNair. 1. Dr. Kinnari Watson 11. Optrins Laboratories Inc.

Introduction

Hydrogen peroxide (H2O2) is primarily associated with cell damage; however, regeneration research shows that cells release low levels of H2O2 as part of normal intercellular high-frequency multiplexing of hydrogen peroxide transport, uptake The mechanisms of release and biological effects are currently unknown, but have important implications for cancer, stem cells, and aging. The doublet red reagent is non-fluorescent based on the oxidation of 10-acetyl 3,7 dihydroxyphenoxazine. The assay principle is simple. It is catalyzed by horseradish peroxidase (HRP), which produces a red fluorescent product in H2O2. Resorcinol, the stoichiometric ratio is 1:1. The final resin product has a high extinction coefficient. This is an ultra-sensitive detection method with only 10 picomoles of H2O2 in a 100 μL volume; 1 × 10–5 U/mL of HRP. These readings can be measured spectrophotometrically or with fluorescence readings using a microplate reader with excitation at 530 nm and emission at 590 nm.

Materials and methods

The OT-2, P300 single GEN2 pipette, and P20 single GEN2 pipette temperature modules were set to 23°C and set to 37°C at the end of the protocol. THP-1 macrophage-like cells (ATCC, Manassas, VA, USA) (No. TIB-202) Phenol red-free RPMI 1640 medium (Thermo Fisher Scientific 11835030) Phorbo 12-myristate 13-acetate ( PMA) (Sigma Aldrich P1585-1MG) Fetal Bovine Serum (FBS) Not heat-inactivated (ATCC, Cat No. 30-2020) Amplex Red Detection Kit (Cat No.A22188), includes a) 5X Reaction Buffer b) HRP c) DMSO d) Amplex Red Reagent e) 3% H2O2 a) Vision Center 96 Filter Tip Holder 200μL b) 96 Filter Tip Rack 20μL c) Vision Center 10 Tube Rack with Falcon 4 x 50 mL, 6 x15mL Conical D) Vision Center 24 Tube Rack with Eppendorf 1.5 mL Safety Lock Cap (Pack of Two)

Conclusion

OT-2 enables automated determination of H2O2 in THP-1 cells. OT-2 can handle medium to high-throughput sample numbers in a 96-well format. OT-2 can simulate manual protocols without loss of time and high reproducibility.

Discuss

This study is to understand the feasibility of automatically detecting H2O2 like Amplex Red in OT-2. H2O2 has been shown to be an important signaling molecule responsible for metabolic activity and aiding in enzyme activation, which can influence cell morphology and cause proliferation in the presence of growth factors. Extracellular H2O2 The cell line used here is human monocytes, THP-1. The results indicate that H production rates in different PMA environments may depend on the concentration of 2O2. The relationship between H2O2 release rate and PMA concentration may be after a significant increase at 10,25 ng/mL compared to the basal rate. OT-2 has been successfully used to automate various experimental protocols in NGS, IP, thereby simplifying the use of standard kits and analytical protocols that can be manually labor-intensive. By automating the analysis steps, consistent detection performance was observed compared to manual protocols. Additionally, this describes the pipetting efficiency between manual and manual protocols for quantifying peroxide/peroxidase. This protocol is capable of giving the user an hour and 11 minutes of hands-on time, which may be useful if multiple 96-well plates of different cell types are required to detect hydrogen peroxide levels with or without the addition of various stimulants or growth factors. Effective method.

Original address: OT-2 THP-1 on the poster Automated determination of cellular hydrogen peroxide levels