Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Summary

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Abstract

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

Introduction

The role of lymphatic vessels and LECs in the liver is not well understood. While lymphatic vessels are found within the portal triad of the liver¹ and expand during disease², very little is understood regarding the function and phenotype of LECs within the liver. With the discovery of markers that are found primarily on LECs³, the importance of these cells within different tissue niches in homeostasis and disease will fill a significant gap in our understanding. LECs have a major role in maintaining peripheral tolerance in the lymph node and in metastatic tumors by interacting directly with T cells^{4,5,6,7,8,9,10,11,12,13}. LECs in the lymph node can promote protective immunity via their interactions with migratory dendritic cells^14,15,16. Therefore, there are multiple roles for LECs which may be specific to the tissues and interactions in which they are present. However, very little is understood about how LECs interact with immune cells in the tissue or how LECs function in different organ systems; thus, evaluating LECs on a per cell basis within the liver or other organs may lead to advances in how LECs program tissue-specific immunity. While much of the literature that focuses on LECs in the liver uses microscopy to visualize LECs using one or two markers and morphology¹⁷, very little has been done to specifically evaluate LECs on a cell by cell basis using flow cytometry, though one study did evaluate differences between liver sinusoidal endothelial cells (LSECs) and LECs¹⁸. Being able to analyze LEC populations in the liver by flow cytometry allows for the in-depth study of LEC phenotype during normal homeostasis or disease.

To evaluate LECs by flow cytometry, multiple surface markers are needed. Typically, LECs are visualized by the expression of prospero-related homeobox 1 (Prox-1), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) or vascular endothelial growth factor receptor 3 (VEGFR3) using microscopy. However, in the liver, the expression of these markers is not restricted to LECs. Prox-1 is widely expressed by hepatocytes during liver development, regeneration, and injury¹⁹, and LYVE1 and VEGFR3 are expressed by the liver sinusoidal endothelial cells¹⁸. In the lymph node, LECs are identified using flow cytometry as clusters of differentiation (CD) CD45-, CD31+, and podoplanin+ (PDPN)¹⁶. However, this approach is too minimal to isolate LECs in the liver since CD45- CD31+ cells will capture endothelial cells, and the predominant population of vascular endothelial cells in the liver are LSECs. Thus, other markers are needed to distinguish the rare LEC population from the abundant LSEC population. Both CD16/32 (expressed by mature LSECs¹⁸) and CD146 (a common vascular endothelial cell marker that is predominately expressed within the liver sinusoids by liver sinusoidal endothelial cells²⁰ with little to no expression by lymphatic endothelial cells²¹) were candidate markers.

Therefore, we optimized a method for isolating and visualizing LECs in the liver using the above markers, CD45, CD31, CD146, CD16/32, and PDPN for flow cytometry. We describe the use of collagenase IV, DNase 1, and mechanical separation for liver tissue digestion into a single-cell suspension. We also describe the use of iodixanol density gradient for the isolation of non-parenchymal cells (NPC) and to eliminate cellular debris. Finally, using multiple markers, we determine the optimal flow cytometry gating strategy to identify LECs from the liver with PDPN as the predominant marker.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus.

1. Preparation of the Materials

1. Make a 5 mg/mL solution of DNase I in phosphate-buffered saline (PBS).
2. Make a digestion mixture by adding 5,000 U/mL of collagenase IV to Click’s EHAA media.
3. Warm the digestion mixture at 37 °C for 30 min prior to use.
4. Make an isolation buffer by adding 4.8% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA) to Hanks’ balanced salt solution (HBSS).
5. Make a red blood cell (RBC) lysis buffer by adding 100 mM ammonium chloride, 10 mM KHCO₃, and 0.1 mM EDTA to distilled H₂O.

2. Preparation of a Single-cell Suspension from a Mouse Liver

1. Euthanize the mouse with CO₂ and cervical dislocation.
2. Spray down the mouse with 70% ethanol to wet its fur. Pin the mouse’s feet to a dissection board.
3. Using dissection scissors to cut the skin about 1 cm above the anus, being careful to cut only through the skin (about 1 mm). Pull the skin away from the body with toothed forceps and insert the scissors between the skin and peritoneum. Open the scissors to separate the skin from the peritoneum and, then, cut the skin from the incision to the neck.
4. Pin the skin to the dissection board using one pin under each arm and above each leg. Pull the peritoneal sac up and cut upwards toward the neck. Grab the lobes of the liver and cut just below the sternum.
   Note: Care should be taken if any of the liver will be used for immunohistochemistry (IHC).
5. Cut around the liver and remove the liver from the mouse and place it in 4 mL of Click’s EHAA media.
6. Using a scalpel, cut the liver in ~1 mm diameter pieces.
7. Add 500 µL of the digestion mixture and 500 µL of the DNase I (2 mg/mL) to the liver.
8. Incubate the liver for 30 min at 37 °C. After 15 min, mix the liquid using a 5 mL pipette.
9. After 30 min of incubation, transfer the digested sample through a 100 µm strainer to a 50 mL conical tube.
10. Gently push the remaining pieces through the filter with the plunger of a 1-mL syringe.
11. Wash the filter with 5 mL of isolation buffer and gently push the tissue through the strainer with the back of a plunger from a 1 mL syringe. Repeat this until the filter is washed with 25 mL of isolation buffer.
12. Centrifuge the cells at 400 x g for 5 min. Carefully aspirate off the supernatant.
13. Resuspend the pellet with 4 mL of RBC lysis buffer. Incubate the cells at room temperature for 5 min.
14. Wash the cells with 10 mL of isolation buffer and centrifuge at 400 x g for 5 min.
15. Count the cells on a hemocytometer to determine the full liver count.
16. Resuspend cells in 5 mL of 20% iodixanol and layer them with 1 mL of PBS.
17. Centrifuge the cells at 300 x g for 15 min without a brake.
18. Remove the layer between the PBS and the iodixanol and place them, through a 100 µm filter, into a new 50 mL conical tube.
19. Wash the cells with 10 mL of isolation buffer and centrifuge at 400 x g for 5 min.
20. Discard the supernatant and resuspend the cells in 500 µL of PBS with 2% fetal bovine serum (FBS).

3. Flow Cytometric Analysis of Single Cells from the Liver

1. Count the cells using a hemocytometer and microscope, using trypan blue exclusion to measure viable cells. Add 10 µL of the cells to 10 µL of trypan blue and immediately place them on a hemocytometer and count the live cells (not blue) under a microscope. Then, calculate the number of cells per microliter.
2. Aliquot approximately 5 million of the remaining nonparenchymal cells into a single well of a 96-well plate.
3. Centrifuge the cells at 400 x g for 5 min.
4. Discard the supernatant and resuspend the cells in 90 µL of PBS with 2% FBS.
5. Add anti-CD45 (1:200), anti-CD146 (1:200), anti-CD31 (1:200), and PDPN (1:200) diluted in 10 µL of 10x 2.4G2 or anti-CD16/32 (1:200).
   NOTE: No Fc block (2.4G2) was used when anti-CD16/32-labeled antibody was used.
6. To determine where positive and negative gates should be set, include a fluorescence minus one (FMO) stain for each color and an isotype control antibody.
7. To determine live versus dead cells, stain with a viability marker (e.g., ghost red 780). Incubate the cells at 4 °C for 30 min.
8. Wash the cells with 100 µL of PBS with 2% FBS.
9. Use a small aliquot of cells to adjust the laser and compensation settings on the flow cytometer. Stain the cells with an antibody to each individual fluorophore and one without any antibody.
   NOTE: Depending on the flow cytometer being used, a compensation matrix should be established to remove spectral overlap.
10. Place the sample tube onto the cytometer probe and collect and record all events.

4. Data Analysis

1. Looking at side-scatter area vs. forward-scatter area, gate on “live” cells based on size and granularity and viability marker dye.
2. Next, using CD45 Brilliant Violet 510 and CD31 PerCp Cy5.5, gate on the CD45- CD31+ cells using the isotype controls and FMO to determine positive and negative populations.
3. Lastly, using CD146 v450 or CD16/32 FITC and PDPN APC, take the CD146- PDPN+ or CD16/32- PDPN+ cells, again using isotype controls and FMO, to determine positive and negative populations. These cells are the LECs.

Results

Studies analyzing liver lymphatics have primarily used immunohistochemistry to quantitate the frequency and diameter of lymphatic vessels in the liver. However, this method does not allow for the evaluation of LECs on a cell-by-cell basis or for expression of multiple markers, cytokines, chemokines, or transcription factors. Therefore, we asked whether liver LECs could be isolated from the liver and evaluated using flow cytometry. Previous work isolating lymph node LECs was performed usin...

Discussion

The overall importance of LECs in immune homeostasis and regulation has recently come to light²⁵. Much of the published lymphatic literature focuses on skin and lymph nodes; however, lymphatics are found throughout the body²⁶ and, thus, our understanding of their importance in different organs is needed. Here we show a method in which LECs in the liver can be studied on a cell-by-cell basis to better understand their concurrent expression of different surface markers, cytok...

Materials

Name	Company	Catalog Number	Comments
Clicks/EHAA media	Irvine Scientific	9195
Collagenase IV	Worthington Biochemical corporation	LS004188
DNase I	Worthington Biochemical corporation	LS002145	Deoxyribonuclease 1
OptiPrep	Sigma Aldrich	D1556	Density Gradient Medium
V450 anti mouse CD146(clone ME-9F1)	BD biosciences	562232
FITC anti mouse CD146 (clone ME-9F1)	Biolegend	134706	Fluorescein isothiocyanate (FITC)
Pacific Blue anti mouse CD31(clone 390)	Biolegend	102422
PerCp/Cy5.5 anti mouse CD31(clone 390)	Biolegend	102420	Peridinin-chlorophyll proteins-Cyanine 5.5 (PerCP-Cy5.5)
APC anti mouse PDPN (clone 8.1.1)	Biolegend	127410	Allophycocyanin (APC), podoplanin (PDPN)
APC/Cy7 anti mouse CD45 (clone 30-F11)	Biolegend	103116
Brilliant Violet 510 anti mouse CD45 (clone 30-F11)	Biolegend	103138
FITC anti mouse CD16/32 (clone 93)	Biolegend	101306	Fluorescein isothiocyanate (FITC)
PerCp/Cy5.5 anti mouse CD16/32(clone 93)	Biolegend	101324	Peridinin-chlorophyll proteins-Cyanine 5.5 (PerCP-Cy5.5)
ghost red 780 viability dye	TONBO biosceinces	3-0865-T100
APC syrian hamster IgG (clone SHG-1)	Biolegened	402102
PerCp/Cy5.5 rat IgG2a (clone RTK2758)	Biolegend	400531
FITC rat IgG2 (clone eBR2a)	ebioscience	1-4321-80
Anti mouse LYVE1 (clone 223322)	R&D systems	FAB2125A
anti-mouse Cytokeratin(clone EPR17078)	abcam	ab181598
anti-mouse F4/80 (clone Cl:A3-1)	Bio-rad	MCA497
BSA (fraction V)	Fischer	BP1600-100	Bovine Serum Albumin (BSA)
Goat serum	Jackson Immunoresearch	017-000-121
Donkey Serum	Jackson Immunoresearch	017-000-121
EDTA	VWR	E177	Ethylenediaminetetraacetic acid (EDTA) -for RBC lysis buffer
Ammonium Chloride	Fischer	A687-500	for RBC Lysis buffer
Potassium Bicarbonate	Fischer	P184-500	for RBC Lysis buffer
Scalpel	Feather	2975#21
100 μm cell strainer	Fischer	22363549
2.4G2	in house/ATCC	ATCC HB-197	FC block to inhibit non-specific binding to Fc gamma + cells -made from hybridoma
Phosphate Buffered Saline (PBS)	Corning	21-040-CV
Hanks Balanced Salt Solution (HBSS)	Gibco	14185-052
Fetal Bovine Serum (FBS)	Atlanta biologicals	S11550
96 well plate	Corning	3788
6 well plate	Corning	3506
50 mL conical	Truline	TR2004
15 mL conical	Falcon	352196
1 mL Pipete tip	USA scientific	1111-2721
200 µL pipete tip	USA scientific	1110-1700
10 µL pipete tip	USA scientific	1111-3700
seriological 10 mL pipete	greiner bio-one	607107
seriological 5 mL pipete	greiner bio-one	606107
Cell incubator	Fischer		Heracell 160i
BD FacsCanto II flow cytometer	BD biosciences
Clinical Centrifuge	Beckman coulter		model X-14R