TITLE: Flow cytometry based monocyte adhesion assay for quantification of endothelial activation in vitro
AUTHORS: Vinnyfred Vincent, Himani Thakkar, Anjali Verma, Atanu Sen, Nikhil Chandran, Archna Singh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Endothelial pro inflammatory activation is a key event in the development of atherosclerosis. In order to study modulation of endothelial activation, quantification of the same in vitro is necessary. At functional level endothelial activation is quantified using monocyte adhesion assay. This involves addition of fluorescently labelled monocytes on top of cultured endothelial cells and quantifying the number of monocytes adhered. Currently this is done using microscopy. He we present a novel flow cytometry based monocyte adhesion assay with clear advantages over previously described methods. </div></div>

[STEPS]
?. [Preparation of human umbilical vein endothelial cell (HUVEC) monolayer ]
Add 1 ml of 0.2% gelatin in PBS to each well of a 6 well plate and keep inside a CO2 incubator at 37oC for 1 hour.
?. [Fluorescent labeling of THP1 monocytes ]
THP1 cells are cultured in RPMI 1640 medium with 10% FBS and antibiotics. Count THP1 cells using a hemocytometer. 5x105  THP1 cells per well are need for the assay. Count and aliqote the total number of cell needed for the assay into a centrifuge tube including 105 cells need for positive gating control during flow cytometry.
?. [Monocyte adhesion under static conditions]
Add 5x105 THP1 cells per well on top of the HUVEC monolayer and keep inside CO2 incubator at 37oC and 5% CO2 for 30 minutes. Keep 105 THP1 cells apart as positive gating control for flow cytometry analysis later.
?. [Preparation of human umbilical vein endothelial cell (HUVEC) monolayer ]
After 1 hour, discard the gelatin and wash with 3 ml PBS. Discard PBS
?. [Preparation of human umbilical vein endothelial cell (HUVEC) monolayer ]
Seed 104 HUVECs/cm2 in each well including a well for negative gating during flow cytometry, in ECGM supplemented with growth factors, 10% fetal bovine serum (FBS) and antibiotics. Keep inside CO2 incubator at 37oC and 5% CO2 till confluent. HUVECs grow as adherent monolayer.
?. [Fluorescent labeling of THP1 monocytes ]
Stock solution of DIL is made in DMSO at 1 mg/ml. This is added to the THP1 cells in complete media (RPMI with 10% FBS) at 1:1000 dilution with the final concentration in the media of 1 ug/ml. After adding DIL keep the THP1 cells inside CO2 incubator at 37oC and 5% CO2 for 30 minutes.
?. [Fluorescent labeling of THP1 monocytes ]
After 30 minutes, centrifuge at 300 g for 5 minutes at room temperature (RT). Discard the supernatant without disturbing the pellet and resuspend in complete media. Repeat this step once more to remain any unbound DIL.
?. [Monocyte adhesion under static conditions]
After 30 minutes remove unbound monocytes by washing with PBS thrice.
?. [Quantification of adhesion using flow cytometry ]
Dislodge HUVECs and bound THP1 cells using TrypLE Express dissociation reagent. Add 500 ul of TrypLE to each well and incubate at 37oC till cells dislodge. Once the cells have dislodged, add 500 ul of complete ECGM to inactivate TrypLE.
?. [Quantification of adhesion using flow cytometry ]
Transfer to microcentrifuge tubes and centrifuge at 300 g for 5 minutes at room temperature (RT). Resuspend the pellet in PBS and repeat the PBS wash once. Resuspend in 500 ul of PBS.
?. [Quantification of adhesion using flow cytometry ]
Run pure populations of HUVECs and THP1 cells for setting the vertical gate for analysis. Set a common gate for both HUVECs and THP1 should be used in FSC vs SSC graph. acquire 104 events in all tubes. Calculate the percentage of cells on either side of the vertical gate to calculate the number of DIL negative and DIL positive cells. DIL negative cells represent HUVECs and DIL positive cells represent THP1 cells.
?. [Quantification of adhesion using flow cytometry ]
Calculate the average number of HUVECs adhered to single HUVEC by dividing the number of DIL positive cells out of the 104 cells with DIL negative cells.