PGEX2-T based β defensin primer designing and isolation of neutrophil, macrophage, bone marrow and mesenchymal stem cells

Abstract

Defensins are antimicrobial peptides that act mainly by disrupting the structure of bacterial cell membranes and are found in many compartments of the body. Defensins play a central role in defense against pathogens and they are considered part of the innate immune response. Material and methods-Reagents: Animal cell culture media - Growth medium D-MEM, RPMI and alpha -MEM was purchased from gibco, CA, USA. Fetal bovine serum (FBS)was obtained from Invitrogen, CA, USA. DAPI, agarose was purchased from SRL, India. Penicillium streptomycin solution was obtained from himedia, Mumbai, India. Animal modal: In bred populations of Swiss albino mice of either sex and at 6-8 weeks of age were used. Mice obtained from the Animal House, All India Institute of Medical Science, NewDelhi-110029 and India Standardization, Isolation and purification and characterization of bone marrow from Swiss albino mice. Bone marrow is consisting of multiple type of stem cells such as -Mesenchymal stem cells (MSCs) which are multipotent stem cells that have the potential to self-renew and differentiate into a variety of specialized cell types such as osteoblasts, chondrocytes, adipocytes, and neurons. Result: Cells were harvested by adherence purification, the adherence of cells which is a symbol of macrophage and acentric nucleus is visible in DAPI stain. The extended cytoplasmic surface and CD14 positive staining show the purified macrophage as 98% purity. BM-MSC were harvested by adherence purification, the adherence of cells to the plastic culture dish surface is a symbol of Mesenchymal stem cell and nucleusis visible in DAPI stain. Conclusion: This procedure is widely used to study macrophage biology since moderate numbers of resident, unstimulated macrophages can easily be obtained fromthe peritonealcavity.