Computational protocol: Effects of Wharton’s jelly-derived mesenchymal stem cells on neonatal neutrophils

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Protocol publication

[…] PMN were incubated with WJ-MSCs (WJ-MSC + PMN, 1:20) or medium control (PMN) for 24 hours, in the presence or absence of lipopolysaccharide (1 μg/mL) and antibodies to ICAM-1 or IL-6 (1 μg/mL). Neutrophils and WJ-MSCs were separated after co-culture by adherence. Nonadherent neutrophils were harvested from the co-culture supernatant, and their purity was confirmed by morphology. Viable WJ-MSCs were identified as adherent to the culture plate after two washes with phosphate-buffered saline. Absence of residual neutrophils and WJ-MSC morphology were confirmed by microscopy. Total RNA was extracted from neutrophils or WJ-MSCs, complementary DNA generated, and gene expression quantified by real-time polymerase chain reaction using RT2 qPCR master mix (Qiagen) and amplified using a Stratagene MX300p instrument, with β-actin as standard. Full-length coding sequences were obtained from GenBank™. Primers were designed using Primer Express software. Forward and reverse primers used were: NOX-1, 5′-CCTTGCACCGGTCATTCTTT-3′ and 5′-CGGTAAAACCGGAGGATCCT-3′; COX-2, 5′-GCCTGATGATTGCCCGACT-3′ and 5′-GCTGGCCCTCGCTTATGATCT-3′; HO-1, 5′-GCTCAAAAAGATTGCCCAGA-3′ and 5′-GCGGTAGAGCTGCTTGAACT-3′; catalase, 5′-CGGAGATTCAACACTGCCAA-3′ and 5′-GAATGCCCGCACCTGAGTAA-3′; VEGF, 5′-GGCGTCGCACTGAAACTTTT-3′ and 5′-TCCGAAGCGAGAACAGCC-3′; TLR4, 5′-CCAGAGCCGCTGGTGTATCT-3′ and 5′-AACTGCCAGGTCTGAGCAATCT-3′; β-actin, 5′-AAAGACCTGTACGCCAACAC-3′ and 5′-GTCATACTCCTGCTTGCTGAT-3′. [...] Data were analyzed using Statistica version 5.5 (StatSoft, Inc., Tulsa, OK, USA). The effects of treatments were compared pairwise by Student’s t-test. […]

Pipeline specifications

Software tools Primer Express, Statistica
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Chemicals NADP