FSP1 Inhibitor Assay Kit

FSP1 (Ferroptosis Suppressor Protein 1), also known as AIFM2, is a NAD(P)H-dependent oxidoreductase localized to the plasma membrane. Studies have shown that the expression level of FSP1 is positively correlated with cancer cell resistance to ferroptosis inducers (such as the GPX4 inhibitor RSL3). This characteristic makes FSP1 a novel target for anticancer therapy, particularly in tumors with GPX4 inactivation or resistance, where targeted inhibition of FSP1 can synergistically induce ferroptosis. Currently, the development of FSP1 inhibitors and their combination therapies with ferroptosis inducers has become a research hotspot in the field of cancer treatment.
The FSP1 inhibitor detection kit employs a fluorescence-based method to measure FSP1 inhibitor activity. Through enzyme-catalyzed reactions with specific substrates, it precisely determines inhibition efficiency, offering advantages such as high sensitivity, ease of operation, and compatibility with high-throughput screening, making it suitable for the screening of FSP1 inhibitors.

The composition and specifications of the FSP1 inhibitor detection kit are as follows:

1. Reagent Preparation
1) Thaw the 10× Detection Buffer at room temperature. Dilute it to 1× with deionized water at a 1:10 ratio. It is recommended to use the diluted buffer on the same day. The undiluted detection buffer can be stored at 4℃ for 1 month.
2) Thaw Resazurin at room temperature. Use it directly (for 96-well plates) or dilute it 1:1 with 1× Detection Buffer (for 384-well plates). Use as soon as possible after thawing; it remains stable for up to 2 h at room temperature protected from light.
3) NADH is provided as a lyophilized powder. Dissolve each vial with 2 mL (for 96-well plates) or 4 mL (for 384-well plates) of 1× Detection Buffer. Use as soon as possible after reconstitution; it remains stable for up to 2 h at room temperature protected from light.
4) Thaw FSP1 on ice. Mix 100 μL of FSP1 with 1900 μL of 1× Detection Buffer (for 96-well plates) or 50 μL of FSP1 with 1950 μL of 1× Detection Buffer (for 384-well plates). It is recommended to prepare fresh and use immediately. Store the diluted solution on ice and use within 2 h. Store undiluted FSP1 at -80 ℃ and avoid repeated freeze-thaw cycles.
5) Thaw the FSP1 Inhibitor at room temperature. Use it directly (for 96-well plates) or dilute it 4-fold with 1× Detection Buffer (for 384-well plates). Use as soon as possible after thawing. Store unused FSP1 Inhibitor at -20 ℃ and avoid repeated freeze-thaw cycles.

2. Procedure
1) Perform the reaction in a 96-well or 384-well black plate. The recommended reaction volume is 100 μL for 96-well plates and 25 μL for 384-well plates.
2) Set up Background wells, 100% Activity wells, Positive Control wells, and Test wells:
① Background wells: Add 75 μL / 10 μL (96-well plate / 384-well plate) of 1× Detection Buffer to each well, followed by 5 μL of the solvent used to dissolve the inhibitor.
② 100% Activity wells: Add 65 μL / 5 μL (96-well plate / 384-well plate) of 1× Detection Buffer to each well, followed by 10 μL / 5 μL (96-well plate / 384-well plate) of FSP1 and 5 μL of the solvent used to dissolve the inhibitor.
③ Positive Control wells: Add 65 μL / 5 μL (96-well plate / 384-well plate) of 1× Detection Buffer to each well, followed by 10 μL / 5 μL (96-well plate / 384-well plate) of FSP1 and 5 μL of the FSP1 Inhibitor provided in the kit.
④ Test wells: Add 65 μL / 5 μL (96-well plate / 384-well plate) of 1× Detection Buffer to each well, followed by 10 μL / 5 μL
(96-well plate / 384-well plate) of FSP1 and 5 μL of the test inhibitor.
3) After addition, gently shake for 20 s, and incubate at room temperature (22~25℃) protected from light for 10 min.
4) Add 10 μL / 5 μL (96-well plate / 384-well plate) of Resazurin to each well, followed by 10 μL / 5 μL (96-well plate / 384-well plate) of NADH to each well. Gently shake for 20 s, and incubate at room temperature (22~25℃) protected from light for 15 min.
5) Read the plate using an excitation wavelength of 540 nm and an emission wavelength of 590 nm.
3. Calculation of Results

Note:
A: Signal of 100% Activity wells — Signal of Background wells
B: Signal of Test wells — Signal of Background wells

1) After first use, the reagent should be aliquoted and stored at -20°C or below, protected from light to ensure stability. 2) The organic solvents used to dissolve inhibitors may affect reaction signals. It is recommended to maintain final concentrations of ≤5% for DMSO, ≤1% for DMF, and ≤1% for methanol or ethanol in the reaction. 3) Mixing different batches is not recommended. Unless validated, altering the reagent quantities in the reaction is not advised. 4) This product is intended for research use only.