Quantification of total and phosphorylated STAT3 by calibrated western blotting.

Quantification of intracellular proteins is essential to understand signaling. Here, we describe quantification of the expression and phosphorylation of the transcription factor STAT3. We present isolation of total and phosphorylated STAT3 from cell lysates by immunoprecipitation, followed by SDS-PAGE and western blot together with known amounts of a calibrator protein that shares an epitope with the precipitated proteins. Finally, we explain how to relate the amount of precipitated protein to the amount of calibrator protein considering the efficiency of immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Dittrich et al. (2012)1 and Reeh et al. (2019).2.

Quantification of membrane-bound cytokine receptors by calibrated flow cytometry.

We present a protocol for quantifying the expression of the receptor gp130 using a calibrated flow cytometric approach. We describe pitfalls for receptor quantification such as titration of primary antibodies and standardizing cell culture. Receptors are stained with primary antibodies and fluorophore-coupled secondary antibodies. Beads covered with defined numbers of immunoglobulin G stained with fluorophore-coupled secondary antibodies serve as calibrators. In this way, the fluorescence intensity of cells is converted to the number of receptors on the cell surface. For complete details on the use and execution of this protocol, please refer to Reeh et al. (2019).1.

Serum soluble Fas ligand is a severity and mortality prognostic marker for COVID-19 patients.

Finding cytokine storm initiator factors associated with uncontrolled inflammatory immune response is necessary in COVID-19 patients. The aim was the identification of Fas/Fas Ligand (FasL) role in lung involvement and mortality of COVID-19 patients. In this case-control study, mild (outpatient), moderate (hospitalized), and severe (ICU) COVID-19 patients and healthy subjects were investigated. RNA isolated from PBMCs for cDNA synthesis and expression of mFas/mFasL mRNA was evaluated by RT-PCR. Serum sFas/sFasL protein by ELISA and severity of lung involvement by CT-scan were evaluated. Also, we docked Fas and FasL via Bioinformatics software (in silico) to predict the best-fit Fas/FasL complex and performed molecular dynamics simulation (MDS) in hyponatremia and fever (COVID-19 patients), and healthy conditions. mFasL expression was increased in moderate and severe COVID-19 patients compared to the control group. Moreover, mFas expression showed an inverse correlation with myalgia symptom in COVID-19 patients. Elevation of sFasL protein in serum was associated with reduced lung injury and mortality. Bioinformatics analysis confirmed that blood profile alterations of COVID-19 patients, such as fever and hyponatremia could affect Fas/FasL complex interactions. Our translational findings showed that decreased sFasL is associated with lung involvement; severity and mortality in COVID-19 patients. We think that sFasL is a mediator of neutrophilia and lymphopenia in COVID-19. However, additional investigation is suggested. This is the first report describing that the serum sFasL protein is a severity and mortality prognostic marker for the clinical management of COVID-19 patients.