Isolation of B-cells using Miltenyi MACS bead isolation kits.

Dannielle K Moore,Ayanda N Shabangu,André G Loxton,Nelita Du Plessis,Bongani Motaung,Jeffrey Chalmers

doi:10.1371/journal.pone.0213832

Dannielle K Moore, Ayanda N Shabangu + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0213832

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Abstract

This article describes the procedures used to isolate pure B-cell populations from whole blood using various Miltenyi magnetic-activated cell sorting (MACS) bead Isolation kits. Such populations are vital for studies investigating the functional capacity of B-cells, as the presence of other cell types may have indirect effects on B-cell function through cell-cell interactions or by secretion of several soluble molecules. B-cells can be isolated by two main approaches: 1) Negative selection—in which B-cells remain “untouched” in their native state; this is advantageous as it is likely that B-cells remain functionally unaltered by this process. 2) Positive selection–in which B-cells are labelled and actively removed from the sample. We used three Negative B-cell isolation kits as well as the Positive B-cell isolation kit from Miltenyi and compared the purity of each of the resulting B-cells fractions. Contamination of isolated B-cell fractions with platelets was the conclusive finding for all of the isolation techniques tested. These results illustrate the inefficiency of current available MACS B-cell isolation kits to produce pure B-cell populations, from which concrete findings can be made. As such we suggest cell sorting as the preferred method for isolating pure B-cells to be used for downstream functional assays.

Highlights

The immune system consists of a collection of cell types responsible for maintaining our health by fighting off infection, eradicating foreign materials and battling disease [1]
The desired purity of a cell-type within an isolated sample equates to 90% or more of the total cell population
It should be highlighted that the acceptable > 90% isolated cell purity stated in the data sheet of the tested isolation kits is only achieved when examining the cellular content of the lymphocyte population, while excluding all cell debris from analysis (Fig 1A)

Summary

Introduction

The immune system consists of a collection of cell types responsible for maintaining our health by fighting off infection, eradicating foreign materials and battling disease [1]. This paper reviews the ability of various B-cell isolation kits available from Miltenyi to isolate pure B-cell populations from human blood, based on the purity of the obtained sample fractions. This isolation method is sufficient for studies only focusing on cell surface receptor expression analysis but not functional downstream experiments, due to the presence of cellular contaminants within the final sample fraction.

Results

Conclusion