TITLE: Assaying NLRP3-mediated LDH and IL-1β release
AUTHORS: Harper JW, Louis R R Hollingsworth

[DESCRIPTION]
A general protocol for assaying NLRP3 activation through the downstream release of LDH and IL-1β from cells. Included are protocol for iBMDMs and THP-1 macrophages.

[STEPS]
SECTION: Overview
1. Optimize these protocol for your particular cells lines, treatment regimens, assay kits, etc. on the smaller scale with wild-type (and control NLRP3-KO, if on hand) cells prior to proceeding with large scale experiments. The sensitivity of the particular plate readers you have available is an important factor here, too.
SECTION: Overview
2. Grow iBMDMs (immortalized bone marrow derived macrophages) to full confluency in a 10-cm plate--one per genotype. Cells are maintained in complete DMEM (DMEM + GlutaMAX‱ + Penicillin-Streptomycin + 10% FBS).
SECTION: Overview
3. Trypsinize the cells, neutralize the trypsin with 5X medium, and resuspend cells thoroughly in a 50-mL falcon tube.
SECTION: Overview
4. Count the resuspended cells, and then seed 200,000 cells in each well of a 96-well plate, in 200 μL of complete DMEM. To use a multichannel pipette, resuspend the cells at 1 M/mL and multichannel 200 μL of the suspension into wells.
 

SECTION: Overview
5. The next day, carefully exchange cells into phenol red-and serum-free DMEM ± LPS (1 μg/mL, 100 μL). 
 

SECTION: Overview
4.1. Here's an example of cell seeding (above) with the genotypes "WT", "FAPP1g1", and "FAPP1g2". The treatments are listed below (UT = untreated, Lysis = Lysis buffer total LDH release control). "Medium only" controls would go in row A, columns 10-12, on the day of the assay.

 

SECTION: Overview
6. Resuspend compounds or lysis buffer to 2X the desired final concentration in phenol red-and serum-free DMEM. For all wells that were treated with LPS, resuspend compounds in phenol red-and serum-free DMEM + LPS (1 μg/mL). Add 100 μL to each well with a multichannel pipette and tap to mix.

 

SECTION: Overview
5.1. Add 100 μL of medium alone to 3 wells. Return plate to the incubator for 4 h.
SECTION: Overview
6.1. Add another 100 μL of medium alone to the set of 3 wells from step 5.1. Place the plate back into the cell culture incubator.
SECTION: Overview
7. When the treatment(s) have run their course, tap the plate to mix, then centrifuge the plate (1000 g, 5 min) to pellet any cells that have lifted.
SECTION: Overview
8. Carefully transfer 150 μL of the supernatant to a new 96-well plate. Excess supernatant can be kept on ice while performing assays for any technical repeats or technical optimization (e.g., if LDH/IL-1β assays are over or under saturated).
SECTION: LDH release from iBMDMs
9. Transfer supernatant (10 μL) and fresh cell culture medium (40 μL) to a 96-well plate assay plate; for example, 96 Well Flat Clear Bottom White Polystyrene Plates (Corning 3610).

 

SECTION: LDH release from iBMDMs
10. Follow the manufacturer's instructions for the CyQUANT‱ LDH Cytotoxicity Assay (Thermo C20300).

SECTION: LDH release from iBMDMs
10.1. The incubation time can be reduced to 10 - 15 min if the wells are approaching saturation. Be sure to wrap the plate in foil during incubation and prior to reading the wells. The plate can be briefly spun without its lid to get rid of bubbles before reading absorbance values.
SECTION: LDH release from iBMDMs
11. Correct all absorbance values (A490 nm - A680 nm).

Calculate % maximum LDH release for each replicate as follows: (Treatment – Average[medium])/(Average[Geneotype-matched maximum lysis control] – Average[medium]).

Where "Treatment" is the absorbance value for the individual treatment replicate; "Average[medium]" is the average absorbance value of the three medium control wells (same for all genotypes); "Average[Geneotype-matched maximum lysis control]" is the average value of the three lysis controls for the specific genotype.

 

SECTION: LDH release from iBMDMs
11.1. This is an example excel file for calculating these values.
  

SECTION: THP-1 macrophages
2. Grow suspension THP-1 monocytes in 10 cm plates between 0.2 - 1 M cells/mL. Cells are maintained in complete RPMI (RPMI + GlutaMAX‱ + 10% FBS + Penicillin-Streptomycin). THP-1s are often cultured without antibiotic, and/or with the addition of BME.
SECTION: THP-1 macrophages
3. Mix and count the suspension cells, and then seed 10,000 cells in each well of a 96-well plate, in 100 μL of complete RPMI. To use a multichannel pipette, resuspend the cells at 0.1 M/mL and multichannel 100 μL of the suspension into wells.

 

SECTION: THP-1 macrophages
3.1. Here's an example of cell seeding (above) with the genotypes "WT", "FAPP1g1", and "FAPP1g2". The treatments are listed below (UT = untreated, Lysis = Lysis buffer total LDH release control). "Medium only" controls would go in row A, columns 10-12, on the day of the assay.

 


SECTION: THP-1 macrophages
6.1. Add 100 μL of medium alone to 3 wells. Return plate to the incubator for 3 h.
SECTION: THP-1 macrophages
4. Add 100 μL of PMA in complete RPMI (50 ng/μL) to each well to differentiate the suspension THP-1 monocytes to adherent THP-1 macrophages. Incubate for 24 h.
SECTION: THP-1 macrophages
5. Carefully exchange the 200 μL medium containing PMA for complete RPMI without phenol red. Incubate for 24 h.

 

SECTION: THP-1 macrophages
6. Carefully exchange the 200 μL complete medium for phenol red-and serum-free RPMI ± LPS (1 μg/mL, 100 μL). 
 

SECTION: THP-1 macrophages
7. Resuspend compounds or lysis buffer to 2X the desired final concentration in phenol red-and serum-free DMEM. For all wells that were treated with LPS, resuspend compounds in phenol red-and serum-free DMEM + LPS (1 μg/mL). Add 100 μL to each well with a multichannel pipette and tap to mix.

 

SECTION: THP-1 macrophages
7.1. Add another 100 μL of medium to the set of 3 wells from step 6.1 (medium background control). Place the plate back into the cell culture incubator.
SECTION: THP-1 macrophages
8. When the treatment(s) have run their course, tap the plate to mix, then centrifuge the plate (1000 g, 5 min) to pellet any cells that have lifted.
SECTION: THP-1 macrophages
9. Carefully transfer 150 μL of the supernatant to a new 96-well plate. Excess supernatant can be kept on ice while performing assays for any technical repeats or technical optimization (e.g., if LDH/IL-1β assays are over or under saturated).
SECTION: LDH release from THP-1s
10. Transfer supernatant (50 μL) to a 96-well plate assay plate; for example, 96 Well Flat Clear Bottom White Polystyrene Plates (Corning 3610).

 

SECTION: LDH release from THP-1s
11. Follow the manufacturer's instructions for the CyQUANT‱ LDH Cytotoxicity Assay (Thermo C20300).

SECTION: LDH release from THP-1s
11.1. The incubation time can be reduced to 15 min if the wells are approaching saturation. Be sure to wrap the plate in foil during incubation and prior to reading the wells. The plate can be briefly spun without its lid to get rid of bubbles before reading absorbance values.
SECTION: LDH release from THP-1s
12. Correct all absorbance values (A490 nm - A680 nm).

Calculate % maximum LDH release for each replicate as follows: (Treatment – Average[medium])/(Average[Geneotype-matched maximum lysis control] – Average[medium]).

Where "Treatment" is the absorbance value for the individual treatment replicate; "Average[medium]" is the average absorbance value of the three medium control wells (same for all genotypes); "Average[Geneotype-matched maximum lysis control]" is the average value of the three lysis controls for the specific genotype.

 

SECTION: LDH release from THP-1s
12.1.   Here is a mock excel spreadsheet for calculating these values 

SECTION: IL-1β release from iBMDMs
9. Dilute supernatant 10X, then add 12.5 μL to a 384-well plate compatible with luminescence measurements. For example, a 384-well Low Flange White Flat Bottom Polystyrene Plates (Corning 3570)

 

SECTION: IL-1β release from iBMDMs
10. Proceed with the Lumit‱ IL-1β Mouse Immunoassay, according to the manufacturer's protocols.

 

SECTION: IL-1β release from iBMDMs
11. Plot the standard curve and experimental values in graphpad prism. 
SECTION: IL-1β release from THP-1s
10. Add 12.5 μL to a 384-well plate compatible with luminescence measurements. For example, a 384-well Low Flange White Flat Bottom Polystyrene Plates (Corning 3570)

 

SECTION: IL-1β release from iBMDMs
11.1. Here is an example of setting up the data for linear interpolation in GraphPad Prism.
 
Here is an example after linear interpolation in GraphPad Prism (Analysis -> Interpolate a standard curve -> Third order poynomial):
 

SECTION: IL-1β release from iBMDMs
11.2. Alternative ways to analyze the data are reported in the manufacturer's manual.
SECTION: IL-1β release from THP-1s
11. Proceed with the Lumit‱ IL-1β human Immunoassay, according to the manufacturer's protocols.

 

SECTION: IL-1β release from THP-1s
12. Plot the standard curve and experimental values in graphpad prism. 
SECTION: IL-1β release from THP-1s
12.1. Here is an example of setting up the data for linear interpolation in GraphPad Prism.
 
Here is an example after linear interpolation in GraphPad Prism (Analysis -> Interpolate a standard curve -> Third order poynomial):
 

SECTION: IL-1β release from THP-1s
12.2. Alternative ways to analyze the data are reported in the manufacturer's manual.