Journal: Journal of Cardiovascular Development and Disease

Article Title: The CCR2 + Monocyte Subsets Increase in Obese Boys but Not Girls with Abnormally High Carotid Intima-Media Thickness: A Pilot Study

doi: 10.3390/jcdd9100330

Figure Lengend Snippet: Gating Strategy. We first gated total leukocytes on a time/side scatter density plot and selected the Zombie UV negative cell population for detecting living cells. Next, we gated living cells for singlets on a forward scatter/trigger pulse width density plot. After recognizing cells by size and granularity, we selected monocytes on the HLA-DR gate. Then, we gated monocytes using the rectangular gating strategy on the CD14 + /CD16 + cell population to identify CD14 ++ CD16 − cells as classical monocytes, CD14 ++ CD16 + cells as intermediate monocytes, and CD14 + CD16 + cells as nonclassical monocytes . We obtained the median fluorescence intensity (MFI) for CCR2 by considering both positive and negative cell populations. We got the CCR2 + cell percentage using fluorescence minus one (FMO) control. For each fluorochrome, we used compensation controls through UltraComp eBeads TM (Invitrogen TM , Carlsbad, CA, USA). We analyzed data by the FlowJo 10.0.7 software (TreeStar, Inc., Ashland, OR, USA).

Article Snippet: Next, we transferred leukocytes to an additional 0.6 mL Eppendorf tube at a density of 2 × 10 6 cells to incubate them with anti-CD14-PECy7 (BD Biosciences, Franklin Lakes, NJ, USA), anti-CD16-PECy5 (BD Biosciences, Franklin Lakes, NJ, USA), and anti-CCR2-Alexa Fluor 647 (BioLegend, San Diego, CA, USA) for 20 min at 4 °C in the absence of light.

Techniques: Fluorescence, Software