A high-throughput CAMP assay for the evaluation of the vitamin D-dependent antimicrobial response Nutrients_MDPI UCLA vitaminD antimicrobial infection immune vitamin CRISPR gene
By Tarun Sai LomteMar 16 2023Reviewed by Danielle Ellis, B.Sc. A recent study published in Nutrients developed a novel and high-throughput assay to evaluate cathelicidin antimicrobial peptide .
Vitamin D is crucial for CAMP expression, and deficient levels have been associated with higher rates of pneumonia, influenza, bacterial vaginosis, tuberculosis, and upper respiratory tract infection.Clustered regularly interspersed palindromic repeats /CRISPR-associated protein 9 -mediated knock-in of the mCherry fluorescent gene reporter was performed at the 3’-end of the CAMP gene in the monocyte-macrophage SC cell line.
The researchers identified a monoclonal population of homozygous CAMPmCherry human SC cells . HiTCA and wild-type SC cells were cultured in media for maintenance and periodically tested for viability. HiTCA cells were incubated with human serum and treatment metabolites, such as vitamin D metabolites or ethanol. They were incubated for 24 hours, and high-throughput flow cytometry analysis was performed. WT cells served as negative controls.
Findings The researchers observed a dose-dependent response of HiTCA cells to 25D3. Ethanol- and 1 nM 25D3-treated cells showed baseline expression . This increased to 50% and 100% with 10 nM and 100 nM 25D3, respectively, comparable to the levels observed in cells treated with 1,252D3. This suggests that HiTCA cells could convert the inactive 25D3 to the active 1,252D3.
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