EasySep™ Direct Human Monocyte Isolation Kit

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Subjects reported to the laboratory following an overnight fast and had forearm vein blood collected into a 10 ml K2EDTA vacutainer tube.

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Subjects reported to the laboratory following an overnight fast and had forearm vein blood collected into a 10 ml K2EDTA vacutainer tube.

Publication protocol

"Subjects reported to the laboratory following an overnight fast and
had forearm vein blood collected into a 10 ml K2EDTA vacutainer tube.
Classical monocytes were isolated from whole blood by column-free
immunomagnetic negative selection using a commercially-available kit
(EasySep Direct Human Monocyte Isolation Kit, StemCell Technologies,
Vancouver, CAN) and associated magnet system (EasyEights EasySep
Magnet, StemCell Technologies) and counted using a Scepter 2.0 automated
cell counter (EMD Millipore, Billerica, MA) with 40 μm sensor.
The kit included CD16 depletion and thus isolated only classical
monocytes (Fig. 1A). For data analysis of assays involving monocyte
isolation, data from one aged subject were excluded on the basis of low
monocyte purity (72%), bringing the total subject number in that group
to 8 for those assays. Inclusion of this subject did not affect the results.
Monocyte purity was established by percent of cells positive for CD14
(anti-CD14-PE, BD Biosciences, San Diego, CA) with analysis on an
Attune NxT flow cytometer (Thermo Fisher Scientific, Grand Island,
NY) (Fig. 1B). Purity of isolated monocytes was tested on all but one
young subject at least once during the study and did not differ between
aged and young subjects (p=0.609, Fig. 1C). All downstream assays
were immediately performed on freshly-isolated monocytes.
We additionally isolated peripheral blood mononuclear cells
(PBMCs) and assessed monocyte subtypes by CD14 and CD16 (anti-
CD16-BV421, BD Biosciences) for comparison purposes to demonstrate
that the magnetic sorting kit removed intermediate and non-classical
monocytes (Fig. 1A). PBMCs were isolated using SepMate-50 conical
tubes and Lymphoprep (StemCell Technologies) according to manufacturer's
instructions."

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