NETosis Assay Kit #41855

NETosis Assay Kit Protocol

Solutions and Reagents

Supplied Reagents

1. PMA (1 mM) Assay Reagent: #51640
2. S7 Nuclease Assay Reagent: #60484
3. EDTA (500 mM) Assay Reagent: #79598
4. NET Assay Neutrophil Elastase Substrate (#34136): The vial contains 15 mM N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide as a substrate for neutrophil elastase.
5. Human Neutrophil Elastase Assay Reagent (#42211): This vial contains human neutrophil elastase at 18 U/mL. To use the enzyme as a positive control, add 2 µL to 2 mL of pre-warmed NET assay buffer. Mix well. Add 100 µL of this diluted enzyme into at least two wells of the assay plate.
6. Bovine Serum Albumin Assay Reagent: #50855
7. Calcium Chloride (1 M) Assay Reagent: #60648
8. 96-Well Solid Plate (Colorimetric Assay): #75820
9. 96-Well Cover Sheet: #87187

NET Assay Buffer (basal medium not included in the kit)

To prepare the NET assay buffer, combine 500 mL of RPMI 1640 base medium (not provided) with 5 g of Bovine Serum Albumin Assay Reagent #50855 and 500 µL of Calcium Chloride (1 M) Assay Reagent #60648. The NET assay buffer is not intended to be sterile and does not need to be prepared or used in a tissue culture hood. Pre-warm the NET assay buffer to 37°C prior to cell stimulation and addition of nuclease to ensure rapid activation and subsequent nuclease activity. For storage of unused NET assay buffer, sterile filter, aliquot, and store at -20°C.

NOTE: Serum contains DNAse that will digest NETs and should be avoided if possible.

Standard Curve

To run a standard curve using the Human Neutrophil Elastase Assay Reagent #42211, obtain eight clean test tubes and label them #1 through #8. NOTE: While the NET assay buffer will serve as an adequate diluent for the Human Neutrophil Elastase Assay Reagent, we recommend adding 400 µL of EDTA (500 mM) Assay Reagent (#79598) to 20 mL of NET assay buffer and using this for the dilutions of the Human Neutrophil Elastase Assay Reagent. Add 5 mL of pre-warmed NET assay buffer into tube #1 and 1 mL into tubes #2-8. Transfer 10 µL of Human Neutrophil Elastase Assay Reagent into tube #1 and mix thoroughly. The concentration of this standard is 36 mU/mL. Serially dilute the standard by removing 1 mL from tube #1 and placing it into tube #2; mix thoroughly. Next remove 1 mL from tube #2 and place it into tube #3; mix thoroughly. Repeat for tubes #4-7. Do not add any standard to tube #8. This tube will be your blank.

Reagent Preparation for treating cells in a 96-well plate (not provided)

1. PMA (1 mM) Assay Reagent #51640 - Prior to use, add 2 µL of PMA (1 mM) Assay Reagent to 1 mL of pre-warmed NET assay buffer to make a 2 µM intermediate stock solution. Subsequently, addition of 40 µL of this intermediate stock solution to 10 mL of pre-warmed NET assay buffer will make a 4X working stock solution.
   1. NOTE: PMA is a potential carcinogen. Wear appropriate protection and use caution when handling this solution.
2. S7 Nuclease Assay Reagent #60484 - Dilute 50 µL of S7 Nuclease Assay Reagent (supplied at 15,000 U/mL) in 20 mL of pre-warmed NET assay buffer immediately prior to use to make a 37.5 U/mL working solution.
3. NET Assay Neutrophil Elastase Substrate #34136 - To assay 80 samples and run a standard curve, dilute 500 µL NET Assay Neutrophil Elastase Substrate into 14.5 mL PBS (a 1:30 dilution).

Reagent Preparation for treating cells in a 24-well plate (not provided)

1. PMA (1 mM) Assay Reagent #51640 - Prior to use, add 1 µL of PMA (1 mM) Assay Reagent to 5 mL of pre-warmed NET assay buffer to make a 10X working stock solution.
   1. NOTE: PMA is a potential carcinogen. Wear appropriate protection and use caution when handling this solution.
2. S7 Nuclease Assay Reagent #60484 - Dilute 12 µL of S7 Nuclease Assay Reagent (supplied at 15,000 U/mL) in 12 mL of pre-warmed NET assay buffer immediately prior to use to make a 15 U/mL working solution.
3. NET Assay Neutrophil Elastase Substrate #34136 - To assay 24 samples in duplicate and run a standard curve, dilute 225 µL NET Assay Neutrophil Elastase Substrate into 6.5 mL PBS (a 1:30 dilution).

Additional Reagents (Not Supplied)

1. RPMI cell culture medium
2. A source of NET-producing cells (e.g., human peripheral blood neutrophils)
3. A 96-well or 24-well tissue culture plate
4. A plate reader with the capacity to measure absorbance at 400-420 nm
5. Phosphate-buffered saline (PBS)

Assay Protocol

Treatments of cells

The following protocol is designed for a 96-well or 24-well tissue culture plate (not provided).

1. Suspend NET-forming cells (e.g., human peripheral blood neutrophils) in pre-warmed NET assay buffer. We recommend a concentration of at least 1 x 10⁶ cells/mL. Add 100 µL of cells per well (96-well) or 800 µL of cells per well (24-well). Be sure to include two wells containing culture medium only for background controls.
2. If working with a 96-well plate, add 50 µL of the 4X working stock solution of PMA or 50 µL of blank NET assay buffer to each well. If working with a 24-well plate, add 100 µL of the 10X working stock solution of PMA or 100 µL of blank NET assay buffer to each well. Incubate at 37°C for four hours.
3. After stimulation and NET formation are complete, gently aspirate the NET assay buffer from the wells and slowly add 100 µL (96-well) or 1 mL (24-well) of pre-warmed NET assay buffer to the sides of the wells. Repeat for a total of two washes. This removes soluble neutrophil elastase that is not NET-associated.
4. If working with a 96-well plate, add 200 µL of the diluted (1:400) S7 Nuclease Assay Reagent to each well. Incubate at 37°C for 45 minutes to disrupt the NETs. If working with a 24-well plate, add 500 µL of the diluted (1:1000) S7 Nuclease Assay Reagent to each well. Incubate at 37°C for 15 minutes to disrupt the NETs.
1. NOTE: For higher cell concentrations, longer incubations (up to one hour) or more S7 nuclease (up to 100 U/mL) may be required.
5. If working with a 96-well plate, add 2 µl of EDTA (500 mM) Assay Reagent to each well to inactivate the nuclease. If working with a 24-well plate, transfer the supernatants to polypropylene microfuge tubes. Add 10 µl of EDTA (500 mM) Assay Reagent to inactivate the nuclease. Centrifuge at 300 x g for five minutes to pellet any cellular debris.
6. Transfer supernatant to a separate 96-well tissue culture plate, new polypropylene microfuge tubes, or other appropriate storage container. Assay for released neutrophil elastase immediately, or store at 4°C for one week or -20°C for up to six months before performing the neutrophil elastase assay.

Performing the Elastase Activity Assay

There is no specific pattern for using the wells on the plate. Each standard and sample should be assayed at least in duplicate.

Use the clear 96-Well Solid Plate (Colorimetric Assay) #75820 included in the kit to perform the assay described below. For optimal results, we recommend pre-warming the standards and samples to 37°C in a water bath prior to performing the NETosis assay.

1. Standard Wells - add 100 µL of standard (tubes #1-8) per well.
2. Sample Wells - Transfer 100 µL of culture supernatant per well.
3. Addition of the Elastase Substrate - add 100 µL of the 1:30 diluted NET Assay Neutrophil Elastase Substrate to each well.
4. Cover the plate with the 96-Well Cover Sheet (#87187) and incubate the plate for 1-2 hours at 37°C.
5. Remove the cover sheet and read the absorbance at 405 nm.

Calculations

Plotting the Standard Curve and Determining the Sample Elastase Activity:

Plot absorbance (linear y-axis) versus concentration (linear x-axis) for standards (S1-S8) and fit the data with a quadratic equation. Using the equation of the line, calculate the elastase activity in each sample. Alternatively, a plot of concentration (y-axis) and absorbance (x-axis) can be performed. This plot has the benefit of easier calculation of elastase activity based on the best-fit quadratic equation.