Quantifying Cytokine Levels via ELISA

ELISA was performed based on the protocol described previously^{10 (link)}. Immunol 2HB-microtiter plates were coated with mouse anti IL-10 (R&D Systems) or mouse anti-IL12 (BD Biosciences) followed by blocking with 2% BSA-PBS for 2 hr. Culture supernatants or diluted sera were added after washing and incubated overnight at 4 °C. Secondary Ab labeled with biotin were added to plates, and incubated for 2 hr. Next plates were developed by phosphatase-conjugated streptavidin (AKP) followed by the addition of p-nitrophenyl phosphatase (pNPP) substrate (Southern Biotech). Optical density was measured using a SpectraMax M5 microplate Reader and SoftMax Pro Acquisition and Analysis software (Molecular Devices).

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Horuluoglu B.H., Kayraklioglu N., Tross D, & Klinman D. (2020). PAM3 protects against DSS-induced colitis by altering the M2:M1 ratio. Scientific Reports, 10, 6078.

Publication 2020

mouse anti IL-10 mouse anti-IL12 phosphatase-conjugated streptavidin p-nitrophenyl phosphatase (pNPP) substrate SpectraMax M5 microplate Reader SoftMax Pro Acquisition and Analysis software

Biotin Devices Elisa Il 10 Il12 Mouse Nitrophenyl phosphatase Optical Phosphatase Pnpp Sera Streptavidin

Corresponding Organization :

Other organizations : National Cancer Institute

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Variable analysis

independent variables

Type of antibody used to coat the microtiter plates (mouse anti IL-10 or mouse anti-IL12)

dependent variables

Optical density (measured using a SpectraMax M5 microplate Reader and SoftMax Pro Acquisition and Analysis software)

control variables

Incubation time (2 hr for blocking, overnight for culture supernatants/diluted sera, 2 hr for secondary Ab)
Blocking solution (2% BSA-PBS)
Wash steps
Addition of phosphatase-conjugated streptavidin (AKP) and p-nitrophenyl phosphatase (pNPP) substrate

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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