EasySep™ Human Monocyte Isolation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For negative selection, PBMCs were diluted to 5 × 107 cells/ml and incubated with 50 µl/ml isolation antibody cocktail and 50 µl/mL platelet removal cocktail for 5 min before the addition of 50 µl/ml RapidSpheres. After an additional 5 min of incubation, PBMCs were placed in the magnet for 3 min and the supernatant, containing monocytes, was collected and washed in PBS (1% FCS).	The monocytes were resuspended in complete maturation medium and placed in the incubator.

Protocol tips
For negative selection, PBMCs were diluted to 5 × 107 cells/ml and incubated with 50 µl/ml isolation antibody cocktail and 50 µl/mL platelet removal cocktail for 5 min before the addition of 50 µl/ml RapidSpheres. After an additional 5 min of incubation, PBMCs were placed in the magnet for 3 min and the supernatant, containing monocytes, was collected and washed in PBS (1% FCS).
Downstream tips
The monocytes were resuspended in complete maturation medium and placed in the incubator.

Publication protocol

"Monocytes were isolated in parallel by either CD14pos selection with EasySep™ Human CD14 Positive Selection Kit II, negative selection with EasySep™ Human Monocyte Isolation Kit (Stemcell Technologies, Vancouver, Canada), or by plastic adherence as described below.

Negative and CD14pos selection
After PBMC isolation, cells were washed again in PBS with 2% FCS and 1 mm EDTA, resuspended in the appropriate buffer, and monocytes were isolated by either negative or CD14pos selection, using immunoselection according to the manufacturer's protocol.

Briefly, PBMCs for CD14pos selection were diluted to 1 × 108 cells/ml and incubated with 100 µl/ml selection antibody cocktail for 10 min before the addition of 100 µl/ml RapidSpheres. After an additional 5 min incubation, PBMCs were placed in the ‘Big Easy’ magnet (StemCell Technologies) for 3 min, the supernatant containing non‐monocyte cells was poured off, and the monocytes were resuspended in PBS (2% FCS, 1 mm EDTA). This was repeated three times. After isolation, CD14pos‐selected monocytes were washed in PBS (1% FCS), and resuspended in complete maturation medium (see below for details), and placed in the incubator.

For negative selection, PBMCs were diluted to 5 × 107 cells/ml and incubated with 50 µl/ml isolation antibody cocktail and 50 µl/mL platelet removal cocktail for 5 min before the addition of 50 µl/ml RapidSpheres. After an additional 5 min of incubation, PBMCs were placed in the magnet for 3 min and the supernatant, containing monocytes, was collected and washed in PBS (1% FCS). The monocytes were resuspended in complete maturation medium and placed in the incubator.

Plastic adhesion
The PBMCs were washed once in PBS with 2% FCS and resuspended in RPMI‐1640 (ThermoFisher Scientific) with 10% human AB serum (Sigma‐Aldrich). For monocyte isolation, 1 × 108 to 2 × 108 PBMCs were plated in 1 Nuclon™ Delta surface treated T‐75 cell culture flasks (ThermoFisher Scientific) at 1 × 107 to 2 × 107 PBMCs/ml in 10 ml, and allowed to adhere in a 5% CO2 container at 37° for 1 hr. Non‐adherent cells were removed by thorough washing with RPMI‐1640. Adherent cells were harvested after 15 min of incubation in PBS with ‘detach buffer’; 0·5% bovine serum albumin, 5 mm EDTA, and 4 mg/ml lidocaine hydrochloride monohydrate (Sigma‐Aldrich) using a cell scraper. Monocytes were washed in PBS with 1% FCS, and resuspended in complete maturation media."

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