Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Isolation, Cell Culture and Cell Stimulation
2.2. Viability Assays
2.2.1. Vitality Staining of Synovial Cells
2.2.2. CellTiter96® Aqueous One Solution Cell Proliferation Assay
2.2.3. CyQUANT® NF Cell Proliferation Assay
2.3. Gene Expression Analysis
2.3.1. RNA Isolation and cDNA Synthesis
2.3.2. qPCR
2.4. Protein Expression Analysis
2.5. Statistical Analysis
3. Results
3.1. Vitality Assays
3.2. DNA Quantification and Cell Proliferation
3.3. Gene Expression
3.4. Protein Synthesis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ACPA | Anti-citrullinated peptide antibody |
bFGF | Basic fibroblast growth factor |
C3aR1 | C3a receptor 1 |
C5aR1 | C5a receptor 1 |
cDNA | Complementary DNA |
CRP | Complement regulatory protein |
CTCF | Corrected total cell fluorescence |
DAPI | 4′,6-Diamidin-2-phenylindol |
DMSO | Dimethyl sulfoxide |
FBS | Fetal bovine serum |
FDA | Fluorescein diacetate |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
h | Hour |
HBSS | Hanks’ balanced salt solution |
hEC | Human endothelial cells |
HSE | Immortalized human rheumatoid arthritic fibroblast-like synoviocytes |
IL | Interleukin |
K4IM | Immortalized human non-arthritic fibroblast-like synoviocytes |
MAC | Membrane attack complex |
MAPK | Mitogen activated protein kinase |
MASP-2 | Mannose-binding protein-associated serine protease 2 |
Min | Minutes |
NF-κB | Nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells |
NP | Nucleocapsid protein |
PAMP | Pathogen-associated molecular patterns |
PBS | Phosphate-buffered saline |
PFA | Paraformaldehyde solution |
qPCR | Real time detection polymerase chain reaction |
RT | Room temperature |
SARS-CoV-2 | Severe acute respiratory syndrome coronavirus type 2 |
TBS | Tris buffered saline |
TCC | Terminal complement complex |
TLR | Toll-like receptors |
TNFα | Tumor necrosis factor |
VEGF | Vascular endothelial growth factor |
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Primer | Company | Sequence | Assay ID | Amplicon Length (bp) |
---|---|---|---|---|
GAPDH | ABI | * | Hs99999905_m1 | 93 |
CD46 | ABI | * | Hs00387246_m1 | 94 |
CD55 | ABI | * | Hs00167090_m1 | 62 |
CD59 | ABI | * | Hs00174141_m1 | 70 |
C3aR1 | ABI | * | Hs00269693_s1 | 82 |
C5aR1 | ABI | * | Hs00704891_s1 | 68 |
TNFα | ABI | * | Hs00174128_m1 | 80 |
IL-6 | ABI | * | Hs00174131_m1 | 95 |
Specificity and Species | Company | Catalogue Number | Stock Concentration | Used Dilution |
---|---|---|---|---|
Mouse anti-human C5aR1 | GeneTex, Eching, Germany | GTX74845 | 1 mg/mL | 1:50 |
Goat anti-human CD55 | R&D systems, Minneapolis, MN, USA | AF2009 | 200 µg/mL | 1:50 |
Rabbit anti-human IL-6 | Novus Biologicals, Centennial, CO, USA | NB600-1131 | + | 1:50 |
Goat anti-human TNFα | Peprotech, Princeton, NJ, USA | 500-P31AG | 0.5 mg/mL | 1:50 |
Mouse anti-human Ki67 | Chemicon International Inc., Temecula, CA, USA | MAB4190 | 1 mg/mL | 1:50 |
Donkey anti-mouse- Alexa Fluor 488 | Invitrogen, Waltham, MA, USA | A21202 | 2 mg/mL | 1:200 |
Donkey anti-goat- Alexa Fluor 488 | Life Technologies, Carlsbad, CA, USA | A11055 | 2 mg/mL | 1:200 |
Donkey anti-rabbit- Alexa Fluor 555 | Life Technologies, Carlsbad, CA, USA | A31572 | 2 mg/mL | 1:200 |
Donkey anti-goat- cyanine (Cy)3 | Dianova, Hamburg, Germany | 705-165-147 | 1.5 mg/mL | 1:200 |
Donkey anti-mouse-Cy3 | Dianova, Hamburg, Germany | 715-166-150 | 1.5 mg/mL | 1:200 |
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Franke, V.; Meyer, S.; Schulze-Tanzil, G.G.; Braun, T.; Kokozidou, M.; Fischlein, T.; Silawal, S. Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα. Life 2022, 12, 1527. https://doi.org/10.3390/life12101527
Franke V, Meyer S, Schulze-Tanzil GG, Braun T, Kokozidou M, Fischlein T, Silawal S. Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα. Life. 2022; 12(10):1527. https://doi.org/10.3390/life12101527
Chicago/Turabian StyleFranke, Vincent, Sophie Meyer, Gundula Gesine Schulze-Tanzil, Tobias Braun, Maria Kokozidou, Theodor Fischlein, and Sandeep Silawal. 2022. "Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα" Life 12, no. 10: 1527. https://doi.org/10.3390/life12101527