Protocols

Method for application of fluid shear stress in microfluidic channels and co-culture assay to determine immunomodulatory potency of mesenchymal stromal cells

Ex vivo culture of cells under precise intensities of shear stress can be accomplished using microfluidic channels in line with fluid pumps. We describe here the protocols used in Diaz et al., Stem Cells, 2017; Kota et al., 2017; and Lee et al., Cellular Signaling, 2017 in a centralized resource with videos, step-by-step instructions, and lists of supplies (Diaz et al., Bio-protocol, 2017).

Application of fluid mechanical force in microfluidics with steady laminar flow (unidirectional recirculation, with minimal pulsatility)

Fluid can be recirculated within microfluidics to apply mechanical force to cultured cells while maintaining minimal pulsatility of flow. In this protocol described in Diaz et al., Methods in Molecular Biology, 2015, we demonstrate that syringe pumps can be used with a series of check valves to limit flow pulsatility that typically occurs with roller-style peristaltic pumps.

Transplantation of neonatal mice with hematopoietic stem cells

A method is described for transplantation of neonatal mice via facial vein to reconstitute the blood system with hematopoietic stem cells from any source. Sublethal irradiation doses are outlined for four strains of mice, including NSG, Rag2 Il2rg double knockout mice, C57BL/6, and B6.SJL (BoyJ).

Data Downloads

Microarray data from Diaz et al., Stem Cells, 2017

GEO Series: GSE82269. Platform: Illumina HumanHT-12 V4.0 expression beadchip. Samples: Human bone marrow mesenchymal stromal cells exposed to static or laminar shear stress conditions. 6 samples in total.

Microarray data from Lee et al., Nature Communications, 2017

GEO Series: GSE73284. Platform: Illumina HumanHT-12 V4.0 expression beadchip. Samples: Human PC3 prostate cancer cells exposed to static or laminar shear stress conditions with or without siRNA knockdown of YAP1. 12 samples in total.

Microarray data from Diaz et al., Journal of Experimental Medicine, 2015

GEO Series: GSE62463. Platform: Illumina MouseWG-6 v2.0 R2 expression beadchip. Samples: Mouse cells derived from the aorta-gonad-mesonephros region exposed to static or laminar shear stress conditions for 6 or 24 hours with or without inhibition of COX2 by indomethacin. 24 samples in total.

Microarray data from Jang et al., Blood, 2015

GEO Series: GSE19951. Platform: Illumina MouseRef-8 v2.0 expression beadchip. Samples: Doxycline-inducible intracellular Notch1 domain (ICN1) murine embryonic stem cells differentiated for 3-5 days with or without induction of Notch1, followed by fluorescence-activated cell sorting for surface expression of VE-cadherin and CD41. 24 samples in total.

Microarray data from Wenzel et al., Developmental Biology, 2011

GEO Series: GSE16533. Platform: Affymetrix Mouse Genome 430 2.0 Array. Samples: Murine lenses collected at embryonic day 17.5 or day of birth (P0) carrying either a wild-type genotype or triply deficient for E2F1, E2F2, and E2F3. 20 samples in total.

Microarray data from Chong et al., Nature, 2009

GEO Series: GSE16454. Platform: Affymetrix Mouse Genome 430 2.0 Array. Samples: Crypts or villi from small intestines with a wild-type genotype or quadruply/triply deficient for Rb, E2F1, E2F2, and E2F3. 24 samples in total.

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