TITLE: TNF ELISA Protocol
AUTHORS: caitlynhenry

[DESCRIPTION]
The purpose of this experiment is to investigate the effects of forskolin-mediated cAMP activation on TNF-ɑ secretion by LPS-treated Schwann cells. The immortalized rat RT4-D6P2T cell line (ATCC #CRL-2768) was cultured and received one of the following treatments: 0.1, 1, or 10 μg/mL of LPS, in N2 media (control) or N2 media supplemented with 2 μM of forskolin, for 3 hours. Cell media samples were collected, and the Rat TNF-alpha ELISA kit (RayBiotech, Cat #ELR-TNFa-1, Norcross, GA) was performed to quantify changes in TNF-ɑ secretion in response to the different treatment combinations. 

[STEPS]
SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
1. Aseptically culture immortalized rat RT4-D6P2T Schwann cells (ATCC, Cat #CRL-2768, Manassas, VA) in Dulbecco's Modified Eagle Medium (DMEM) (ATCC, Cat #30-2002, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher, Cat #16000044, Waltham, VA) and 1% penicillin/streptomycin (Pen-strep) (GIBCO, Cat #15140-015, Gaithersburg, MD)/amphotericin B (R&D Systems, Cat #B23192, Minneapolis, MN) at 37°C and 5% CO2 in poly-L-lysine (PLL)-coated dishes.
SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
2. At 80% confluency, split and seed cells into DMEM (2 mL DMEM/well) in three PLL-coated 6-well plates at a density of ~300,000 cells/well. 
SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
3. Incubate cells in DMEM for 24 hours.
SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
4. After 24 hours, aspirate the DMEM and wash each well 2-3x with 2 mL HBSS. After the last wash, add 2 mL N2 media (DMEM/F12, no phenol red [Thermo Fisher, Cat #21041025, Waltham, MA] supplemented with 5 μg/mL insulin [Sigma, Cat #91077C, St. Louis, MO] and 100 μg/mL apo-transferrin [Sigma, Cat #T1147, St. Louis, MO]) to each well.
SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
5. Incubate cells in N2 media for 24 hours.
SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
6. After 24 hours, prepare the forskolin-supplemented media by adding 10 μL of a 2 mM forskolin stock to 20 mL of N2 media.
SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
7. Add 2 mL of the appropriate medium to each well following the plate layout (see example below).

 

SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
8. After adding the media, add the appropriate LPS dose to each well following the plate layout.

0.1 μg/mL LPS: 2 μL of 100 μg/mL LPS stock OR 20 μL of 10 μg/mL LPS stock

1 μg/mL LPS: 2 μL of 1 mg/mL LPS stock OR 20 μL of 100 μg/mL LPS stock

10 μg/mL LPS: 20 μL of 1 mg/mL LPS stock

SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
9. Allow cells to incubate in the different treatment combinations for the required incubation time (3 hours).
SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
10. After the required incubation time, remove the 6-well plates from the incubator and collect desired volume of media from each well.
SECTION: To prepare RT4-D6P2T cell media samples (for three 6-well plates):
11. Store media samples at -80°C for future use.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
12. Bring all reagents/samples to room temperature.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
13. Make the appropriate volume of 1X Diluent B by adding DI H2O to 5X Diluent B (Item E).
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
14. Standard Preparation:
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
14.1. Gently vortex a vial of Standard Protein (Item C).
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
14.2. Add 400 μL 1X Diluent B directly to Item C and gently vortex to mix.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
14.3. Add 120 μL diluted standard to 880 μL 1X Diluent B.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
14.4. Perform a serial dilution to achieve the required standard concentrations (see example below).

 

SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
15. Sample Preparation:
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
15.1. Dilute samples in 1X Diluent B.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
15.2. For RT4-D6P2T media samples, use a 1:2 or 1:1 dilution.

1:2 dilution: 83.3 μL sample + 166.7 μL 1X Diluent B
1:1 dilution: 125 μL sample + 125 μL 1X Diluent B
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
16. Add 100 μL of each standard and sample into the appropriate wells of the 96-well plate following the plate layout (see example plate layout below).
 
 
 
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
17. Cover the plate and incubate for 2.5 hours on a rocker at room temperature.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
18. While waiting, prepare the desired volume of 1X wash solution by adding DI H2O to 20X wash buffer.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
19. Antibody Preparation:
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
19.1. Gently vortex the detection antibody (Item F).
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
19.2. Add 100 μL 1X Diluent B directly to the vial and gently pipette up and down to mix.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
19.3. Dilute the detection antibody concentrate 80-fold using 1X Diluent B.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
20. After 2.5 hours, discard the solution from each well.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
21. Wash each well 4x with 1X wash solution. After each wash, invert the plate and blot it against a diaper sheet until the diaper sheet becomes dry. After the last wash, aspirate every last drop of wash solution.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
22. Add 100 μL of the prepared detection antibody to each well, cover the plate, and incubate for 1 hour on a rocker at room temperature.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
23. HRP-Streptavidin Preparation:
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
23.1. Gently vortex HRP-Streptavidin Concentrate (Item G) and pipette up and down to mix.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
23.2. Dilute the HRP-Streptavidin Concentrate 200-fold using 1X Diluent B.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
24. After 1 hour, discard the solution from each well and repeat washes following Step 10.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
25. Add 100 μL of the prepared HRP-Streptavidin solution to each well, cover the plate, and incubate for 45 minutes on a rocker at room temperature.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
26. After 45 minutes, discard the solution from each well and repeat washes following Step 10.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
27. Add 100 μL TMB One-Step Substrate Reagent (Item H) to each well, cover the plate completely in aluminum foil, and incubate for 30 minutes on a rocker at room temperature in the dark.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
28. After 30 minutes, add 50 μL Stop Solution (Item I) to each well. Immediately read the plate on the plate reader at 450 nm.
SECTION: To perform TNF ELISA (using RayBiotech Rat TNF-alpha ELISA kit [Cat #ELR-TNFa-1, Norcross, GA]):
29. Plot the standard curve (see example below). Use the standard curve to approximate the concentration of TNF-alpha present in each sample. NOTE: Do not forget to correct for the dilution factor (for example, if you did a 1:1 dilution, multiply the TNF-alpha concentrations by 2).