Dec 20, 2019
Flow cytometry based monocyte adhesion assay for quantification of endothelial activation in vitro
- Vinnyfred Vincent1,
- Himani Thakkar1,
- Anjali Verma2,
- Atanu Sen3,
- Nikhil Chandran1,
- Archna Singh1
- 1Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India;
- 2Department of Zoology, University of Delhi, New Delhi, India;
- 3Department of Cardiac Biochemistry, All India Institute of Medical Sciences, New Delhi, India
Protocol Citation: Vinnyfred Vincent, Himani Thakkar, Anjali Verma, Atanu Sen, Nikhil Chandran, Archna Singh 2019. Flow cytometry based monocyte adhesion assay for quantification of endothelial activation in vitro. protocols.io https://dx.doi.org/10.17504/protocols.io.ban5idg6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 19, 2019
Last Modified: December 20, 2019
Protocol Integer ID: 31165
Keywords: Atherosclerosis, Endothelial cell, Monocyte, Flow Cytometry, Cardiovascular Disorders, Human Umbilical Vein Endothelial Cell, Endothelial dysfunction
Abstract
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.
Guidelines
1. When doing fluorescent labeling of monocytes, any cell impermeable dye can be used. Make sure to use a dye with high stain index so that there is good separation of negative and positive cells.
Materials
MATERIALS
TrypLE™ Express Enzyme (1X), phenol redThermo FisherCatalog #12605010
Dil Stain (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate ('DiI'; DiIC18(3)))Thermo FisherCatalog #D282
Human Umbilical Vein Endothelial Cells (HUVECs)
THP1 monocytes
Endothelial Cell Growth MediaR&D SystemsCatalog #CCM027
RPMI 1640 HimediaCatalog #AS162A
0.2 % gelatin in PBS
Cell culture plates (6 well)
Phosphate buffered saline (PBS) pH 7.4
Safety warnings
Validate that the cell lines are not contaminated with infectious agents, especially if primary human umbilical vein endothelial cells (HUVECs) are isolated in the lab from donors.
Before start
Bring all the reagents to 37oC before starting the assay.
Preparation of human umbilical vein endothelial cell (HUVEC) monolayer
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.
1h
After 1 hour, discard the gelatin and wash with 3 ml PBS. Discard PBS
5m
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.
Overnight
5m
Fluorescent labeling of THP1 monocytes
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.
15m
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.
30m
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.
15m
Monocyte adhesion under static conditions
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.
30m
After 30 minutes remove unbound monocytes by washing with PBS thrice.
15m
Quantification of adhesion using flow cytometry
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.
5m
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.
15m
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.
15m
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.
5m