Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing

Association of mast cells with lung function in chronic obstructive pulmonary disease

BACKGROUND: In asthma, higher chymase positive mast cell (MC-C) numbers are associated with less airway obstruction. In COPD, the distribution of MC-C and tryptase positive mast cells (MC-T) in central and peripheral airways, and their relation with lung function, is unknown. We compared MC-T and MC-C distributions in COPD and controls without airflow limitation, and determined their relation with lung function. METHODS: Lung tissue sections from 19 COPD patients (median [interquartile range] FEV(1)% predicted 56 [23–75]) and 10 controls were stained for tryptase and chymase. Numbers of MC-T and MC-C were determined in different regions of central and peripheral airways and percentage of degranulation was determined. RESULTS: COPD patients had lower MC-T numbers in the subepithelial area of central airways than controls. In COPD, MC-T numbers in the airway wall and more specifically in the epithelium and subepithelial area of peripheral airways correlated positively with FEV(1)/VC (Spearman's rho (r(s)) 0.47, p = 0.05 and r(s )0.48, p = 0.05, respectively); MC-C numbers in airway smooth muscle of peripheral airways correlated positively with FEV(1)% predicted (r(s )0.57, p = 0.02). Both in COPD patients and controls the percentage of degranulated MC-T and MC-C mast cells was higher in peripheral than in central airways (all p < 0.05), but this was not different between the groups. CONCLUSION: More MC-T and MC-C in peripheral airways correlate with better lung function in COPD patients. It is yet to determine whether this reflects a protective association of mast cells with COPD pathogenesis, or that other explanations are to be considered

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference