Optimisation of laboratory procedures for isolating human peripheral blood derived neutrophils.

Functional analysis of neutrophils requires isolation of these cells in the laboratory. Current isolation procedures are time consuming and can potentially activate the resting neutrophils. Thus, in this present study, we have optimised an existing laboratory protocol for human neutrophil isolation from peripheral blood. Twenty ml of blood samples were subjected to optimised density gradient separation and dextran sedimentation to obtain a pure population of neutrophils. The efficacy of the optimised manual post isolation of neutrophils was compared with pre isolation count performed by an automated haematology analyzer. The recovery of neutrophils via our optimised methods was 65.5% in comparison with neutrophils counts at pre-isolation. The morphological analysis of isolated neutrophils indicated the purity level more than 95% using Leishman staining. Our optimised laboratory procedures for neutrophils isolation successfully harvested neutrophils with good viability, purity and post recovery yield. This procedure provides an ideal platform to separate neutrophils for in vitro studies.

Mesenchymal stem cells inhibit proliferation of lymphoid origin haematopoietic tumour cells by inducing cell cycle arrest.

We have previously shown that mesenchymal stem cells (MSC) inhibit tumour cell proliferation, thus promising a novel therapy for treating cancers. In this study, MSC were generated from human bone marrow samples and characterised based on standard immunophenotyping. When MSC were co-cultured with BV173 and Jurkat tumour cells, the proliferation of tumour cells were profoundly inhibited in a dose dependent manner mainly via cell to cell contact interaction. Further cell cycle analysis reveals that MSC arrest tumour cell proliferation in G0/G1 phase of cell cycle thus preventing the entry of tumour cells into S phase of cell cycle.

Cytotoxicity and scanning electron microscopy study of gentamycin-coated HA effect on biofilm.

Biofilms are adherent, multi-layered colonies of bacteria that are typically more resistant to the host immune response and routine antibiotic therapy. HA biomaterial comprises of a single-phased hydroxyapatite scaffold with interconnected pore structure. The device is designed as osteoconductive space filler to be gently packed into bony voids or gaps following tooth extraction or any surgical procedure. Gentamycin-coated biomaterial (locally made hydroxyapatite) was evaluated to reduce or eradicate the biofilm on the implant materials. The results indicated that the HA coated with gentamycin was biocompatible to human osteoblast cell line and the biofilm has been reduced after being treated with different concentrations of gentamycin-coated hydroxyapatite (HA).

Mouse bone marrow mesenchymal stem cells acquire CD45-CD106+ immunophenotype only at later passages.

Classically, MSC are identified by a CD45-CD106+ phenotype. In this study, we found that mouse MSC achieve this characteristic phenotype only at later passages. With increasing passages, CD45 (hematopoietic marker) expression shifts to negativity, whereas CD106 (vascular cell adhesion molecule-1) expression becomes increasingly positive. These results demonstrate that MSC cells cultured from mouse bone marrow acquire a classical MSC immunophenotype (CD45-CD106+) in later passages.

Mouse bone marrow mesenchymal stem cells acquire CD45-CD106+ immunophenotype only at later passages

Classically, MSC are identified by a CD45-CD106+ phenotype. In this study, we found that mouse MSC achieve this characteristic phenotype only at later passages. With increasing passages, CD45 (hematopoietic marker) expression shifts to negativity, whereas CD106 (vascular cell adhesion molecule-1) expression becomes increasingly positive. These results demonstrate that MSC cells cultured from mouse bone marrow acquire a classical MSC immunophenotype (CD45-CD106+) in later passages.

Cytotoxicity and scanning electron microscopy study of gentamycin-coated HA effect on biofilm

Biofilms are adherent, multi-layered colonies of bacteria that are typically more resistant to the host immune response and routine antibiotic therapy. HA biomaterial comprises of a single-phased hydroxyapatite scaffold with interconnected pore structure. The device is designed as osteoconductive space filler to be gently packed into bony voids or gaps following tooth extraction or any surgical procedure. Gentamycin-coated biomaterial (locally made hydroxyapatite) was evaluated to reduce or eradicate the biofilm on the implant materials. The results indicated that the HA coated with gentamycin was biocompatible to human osteoblast cell line and the biofilm has been reduced after being treated with different concentrations of gentamycin-coated hydroxyapatite (HA).