Impact of Different Cell Isolation Techniques on Lymphocyte Viability and Function

Abstract

The outcome of immunological assays is markedly influenced by the method of isolation of lymphocytes. It is, therefore, important to comparatively assess various techniques of isolation of lymphocytes, an aspect thus far not thoroughly addressed. In particular, the potential of isolation techniques to influence cell recovery, viability, and function has not yet been evaluated. These studies were designed to determine the effect of different mechanical tissue dissociation methods on the viability and function of lymphocytes. Following spleen and thymus removal, the lymphoid organs were dissociated by one of four different tissue dissociation techniques: metallic screen, sheer force slide, commercial stomacher, or plunger-screen. Cells were then enumerated and a trypan blue exclusion technique and 7-amino-actinomycin D (7-AAD) were both employed to assess viability. Mitogen-induced lymphocyte proliferation was measured using the Alamar Blue assay. Cell viability and lymphocyte surface antigen expression were assessed using flow cytometry. No significant differences in lymphocyte viability, morphology, or surface antigen expression were observed among the different techniques. Likewise, cellular apoptosis and necrosis were comparable across all the techniques. However, mitogen induced splenic T-cell proliferation was higher in cells collected using the metallic screen and plunger-screen isolation methods as compared to the sheer force slide or commercial stomacher procedures. These data suggest that cell recovery, morphology, and viability are not affected by isolation techniques. However, lymphocyte function, as assessed by mitogen induced proliferation, was negatively affected by the sheer force slide or commercial stomacher isolation techniques.

An enhanced postnatal autoimmune profile in 24 week-old C57BL/6 mice developmentally exposed to TCDD

“…Monoclonal antibodies (mAbs) with phycoerythrin (PE) fluorescent labels were used according to manufacturer (BD Pharmingen; San Diego, CA) recommendation at a concentration of 0.2 µg/µL; mAbs with fluorescein isothiocyanate (FITC) fluorescent labels were similarly used at the recommended concentration of 0.5 µg/µL. Cells were stained as previously described (Gogal et al, 2001;Klein et al, 2006). Briefly, lymphocyte aliquots (5 × 10 5 cells/ 100 µL) from thymus, spleen, lymph node and bone marrow were incubated with the following primary mAbs: PE-anti CD4, FITC-anti CD8, FITC-anti CD25, PE-anti CD45/ B220 (ebioscience, San Diego, CA); FITC-anti Vβ 3 TCR, FITC-anti Vβ17a TCR, FITC-anti CD45/B220, PE-anti CD24 (HSA), FITC-anti CD1, PE-anti CD23 (BD Pharmingen).…”

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

“…Lymphocytes are fragile cells that do not survive for long durations in ex vivo conditions [14][15][16] . This is an even greater issue when they are cultured in suboptimal growth media lacking essential nutrients, such as those used in extracellular flux analysis.…”

Tracking the Elusive Lymphocyte: Methods of Detection during Adoptive Immunotherapy

“…While numerous methods of cell dissociation have been developed including both enzymatic and non-enzymatic preparations, there remains no definitive technique without significant drawbacks. Enzymatic methods of dissociation are associated with altered surface antigen presentation, while non-enzymatic suspensions routinely contain large amounts of aggregates of cells along with dead cells Klein et al, 2006). This section will focus on mechanical methods of cell dissociation from tissue, as it avoids loss of surface antigens and potentially retains greater sensitivity for the detection of AT cells by flow cytometry.…”