Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.
The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.
Monocytes constitute a phagocytic component of innate immunity that is essential for fighting pathogens, cleaning up damaged tissues, angiogenesis, and the pathophysiology of many diseases including cancers1,2,3. Monocytes are bone marrow-derived cells composed of heterogeneous subpopulations that circulate in the blood but can be recruited to the site of inflammation in peripheral tissue through specific molecular mechanisms. The recruitment cascades of monocytes, as for leukocytes in general, implicates different steps including capture, rolling, crawling, arrest, transendothelial migration (transmigration) and migration through the vessel wall (basement membrane and mural cells)4. These steps mainly involve inflammation-induced molecules on the endothelial luminal surface such as selectins, glycoprotein ligands, chemokines, intercellular and junctional adhesion molecules, and their receptors on leukocytes such as selectin ligands and integrins. Trafficking pathways through either the endothelial cell junctions (paracellular) or through the endothelial cell body (transcellular) can be used by leukocytes to cross the endothelial barrier5. Whilst monocytes have historically been documented to transmigrate through the transcellular route, potential divergences in their migratory pathway have been proposed as monocytes are no longer considered a homogeneous cell population. It is now becoming clear that monocyte diversity can be defined by each of their differences and commonalities, with respect to their distinctive extravasation cascades3,6. Therefore, in order to unambiguously discriminate between monocyte subpopulations, it is crucial to visualize and phenotype the behavior of each of these different subpopulations during the recruitment process.
Monocytes from human, pig, rat and mouse were subdivided into phenotypic subpopulations with certain ascribed functions and specific migratory behaviors7,8,9. For example, in humans, monocytes can be divided into three subsets based on their surface expression of CD14, a coreceptor for bacterial lipopolysaccharide, and CD16, the Fc-gamma receptor III. Human monocyte subpopulations include classical CD14+CD16–, intermediate CD14+CD16+ and non-classical CD14dimCD16+ cells6,9. The classical CD14+CD16– monocytes were shown to be mainly inflammatory whereas the pool of CD16+ monocytes were collectively found to present TIE2 expression and proangiogenic function10. Consistently, endothelial cell stimulation with inflammatory cytokines such as human tumor necrosis factor (TNF)α or interleukin (IL-1)beta (conventional inflammation) is sufficient to trigger the complete recruitment of classical CD14+CD16– monocytes. However, simultaneous actions of vascular endothelial growth factor (VEGF)A and TNFα (angiogenic factors-driven inflammation) are required to provoke the transmigration of the CD16+ proangiogenic pool of monocytes3. Historically, the traditional Transwell system under static conditions, the parallel plate flow chamber, and the µ-slide flow chambers have been used to quantitatively analyze the recruitment of one leukocyte population at a time in vitro11,12,13. Whilst these protocols have been validated, a more robust method that allowed the simultaneous analysis of multiple monocyte subpopulations would be considered more insightful. Such methodologies must account for multiple interactions and the differing frequencies of each respective population and also provide a mechanistic understanding of the similarities and specificities for the recruitment cascades that define each monocyte subset.
Here, we present a method based on the time-lapse imaging of monocyte recruitment under flow which allows the migratory cascades of different monocyte subpopulations to be studied simultaneously by using confocal microscopy. This method integrates certain critical features that mimic endothelial cell inflammation, as well as the hemodynamics of circulating monocytes in post-capillary venules, the main location of leukocyte recruitment in vivo. The proposed method uses human umbilical vein endothelial cells (HUVEC), which are generated through a well-established protocol of isolation from human umbilical cords. This clinical resource has the advantage of being easily available as a biological by-product, whilst also providing a reasonable yield of endothelial cells that can be isolated from the umbilical vein. We also used fluorescent dyes and immunofluorescence to distinguish between the different cellular components, and confocal microscopy to unambiguously define monocyte positioning (luminal vs. abluminal) over time. The protocol presented here has been developed to simultaneously measure the transmigration levels of monocyte subpopulations. Moreover, it should be noted that this methodology can be extended to study other leukocytes subpopulations and recruitment processes by use of different biomarkers and labelling.
Human materials were used with the informed consent of volunteer donors and in accordance with the Swiss Ethics Committees on clinical research.
1. Isolation and Freezing of Human Umbilical Vein Endothelial Cells (HUVEC)
2. HUVEC Defrosting
NOTE: Use HUVEC at low passage for experiments (maximum 5 passages).
3. HUVEC Culture in 0.4 µ-Slide Chamber
4. HUVEC Staining for Monocyte Recruitment Assay Under Flow
5. Isolation of Human Pan Monocytes and Staining of Subpopulations
6. Preparation of the Fluidic System
7. Time-lapse Imaging of Monocyte Recruitment Under Flow by Confocal Microscopy
8. Analyze the Data with ImageJ
Determining the state of HUVEC activation induced by TNFα
The bio-activity of the inflammatory cytokine TNFα can be vary according to the batch and the repletion of freezing-thawing cycle. It is important to check the activation status of HUVEC with TNFα treatment. This could be performed by staining in parallel some samples of confluent HUVEC for the inflammatory induction of selectins, ICAM-1 and VCAM-115,16,17. An easier and simpler way to check the activation status of HUVEC after TNFα treatment is the morphological change displayed by endothelial cells under inflammatory stress. As shown in Figure 3, HUVEC elongate after 6-h in the presence of TNFα in comparison to unstimulated cells. Similar elongation is observed when HUVECs are stimulated by a mix of TNFα and VEGFA. Recording the activation status of HUVEC is important as the final results of the transmigration of monocytes will depend on the quality of endothelial cell activation.
Monocyte transmigration makes a characteristic discontinuation in endothelial cells
To study monocyte transmigration under flow, we used confocal microscopy with endothelial cells stained in green with CMFDA and the nuclei of isolated monocytes stained in blue with the cell-permeable Hoechst 33342 dye (Figure 4). The time-lapse confocal imaging allowed the visualization of monocytes at the apical plane, where their phenotype could be assessed (Figure 4A-C, Supplemental Movie 1). Migrating cells undergoing transmigration moved to the intercellular space corresponding to cell-cell junctions before they disappeared from the apical plane and appeared in the basal plane. The transmigrated cells presented a black hole around the nucleus corresponding to the monocyte shapes. This shape constantly changed during monocyte migration underneath endothelial cells (Figure 4A-C, Supplemental Movies 2-3). This dynamic black hole made by the monocyte body underneath the endothelial cells, and the monocyte positioning, allowed for the unambiguous identification of transmigrated cells. Quantitation of monocyte recruitment over time showed monocyte adhesion followed by transmigration (Figure 4D-E). Although leukocytes can extravasate through both the transcellular and paracellular routes, we could only observe the paracellular transmigration under flow with this method. This is consistent with our previous observations3,11,18,19.
Angiogenic factor driven inflammation promotes the transmigration of CD16+ monocytes
By using this method, we analyzed the transmigration of human proangiogenic versus non-angiogenic monocytes through an endothelial monolayer stimulated by the inflammatory cytokine TNFα alone or in combination with the angiogenic factor VEGFA. Human proangiogenic monocytes can be identified by the expression of CD16 or TIE2 on their surface. Here, anti-CD16-PE antibody was used to discriminate between pro- and non-angiogenic monocytes. As shown in Figure 5A-B (Supplemental Movies 4-5), the transmigration rate of CD16+ monocytes was low when endothelial cells were stimulated with TNFα only. However, this rate increased when endothelial cells were stimulated simultaneously with TNFα and VEGFA (Figure 5C-E, Supplemental Movies 6-7). The transmigration rate of non-angiogenic monocytes was similarly high under both inflammatory conditions. For both cell subpopulations, the transmigration occurred exclusively through the paracellular route. This method therefore allows for the transmigration aptitudes of different monocytic populations to be investigated simultaneously.
The purity of monocytes affects the transmigration efficiency
The peripheral blood mononuclear cells are composed of T cells, B cells, NK cells and monocytes. The monocyte isolation method used here requires the depletion of the other leukocyte populations from PBMC. To understand how the lack of monocyte purity affects the results, we used PBMCs and stained for pan-monocytes with an anti-CD14-PE antibody before performing the recruitment assay under flow. As shown in Figure 6, HUVEC stimulation with TNFα or TNFα+VEGFA induced the transmigration of only the monocyte population. The other leukocytes composed of T cells, B cells and NK cells did not transmigrate under TNFα or TNFα+VEGFA. Indeed, it has been documented that these leukocytes need other signals for transmigration. Thus, an inefficient isolation of monocytes will lead to an underestimation of monocyte transmigration, as the other leukocytes would be counted as monocytes. This would lead to an erroneous result on monocyte transmigration, due to the contamination of monocyte population with other leukocytes.
Figure 1: Profiling of isolated monocytes by flow cytometry. (A) Analysis of the morphology of PBMC before lymphocyte depletion. The size (forward scatter: FSC) and granularity (side scatter: SSC) of peripheral blood mononuclear cells were determined by flow cytometry. (B) The size and granularity of isolated monocytes were determined by flow cytometry after lymphocyte depletion. An efficient isolation of monocytes shows a complete depletion of the lymphocyte population. Please click here to view a larger version of this figure.
Figure 2: Diagram of the fluidic system. (A) Schematic overview of the perfusion system before and after connection of the slide and mounting on the syringe pump. (B) Diagram of the process of connecting the slide with the tubing using clamps. Please click here to view a larger version of this figure.
Figure 3: Checking the efficient activation of endothelial cells. The activation of HUVEC by inflammatory stimuli was checked by analyzing the cell shape using phase contrast microscopy. After 6 hours of treatment, HUVEC present an elongated morphology when stimulated with TNFα (500 U/mL) or a mix of TNFα (500 U/mL)+VEGFA (1 µg/mL) compared to unstimulated cells. This morphological change of HUVEC following the inflammatory stimulation is an easy-to-detect indicator of the cell activation, which should be ensured for the flow assay. Scale bar = 120 µm Please click here to view a larger version of this figure.
Figure 4: Identification of the transmigrated monocytes by confocal microscopy. (A) Diagram of monocyte transmigration with the expected views at apical and basal planes. The nuclei of monocytes stained with Hoechst 33342 are depicted in blue, and the theoretical shapes of monocytes are depicted with dashed lines around the nuclei. In the basal view, the transmigrated flat monocytes are shown to occupy a space underneath endothelial cell. This space appears as a black hole surrounding the monocyte nucleus on confocal images. (B) Localization of a monocyte before and after transmigration. The orthogonal views are shown, and the appearance of a black hole (delineated with the white dashed line) can be observed after monocyte migration to the endothelial abluminal compartment. A red arrowhead indicates the position of a monocyte before transmigration and the white arrowhead indicates the same cell after transmigration. The orthogonal views show that the transmigrated monocyte is underneath the endothelial cell. Scale bar = 40 µm. (C) Time-lapse image sequences (from 0 to 20 min) of monocyte recruitment overtime. The apical and basal views are shown. The full sequences can be seen in Supplemental Movies 1, 2 and 3. Red squares highlight a transmigrated monocyte with a blue nucleus. The black hole corresponding to the flat body of the monocyte underneath the endothelial cell is delineated by a dashed yellow line. Scale bar = 40 µm. (D) Quantification of monocyte adhesion to TNFα-stimulated versus unstimulated HUVEC over time. (E) Quantification of monocyte transmigration rate over time. N = 3 biological replicates. Data are presented as mean ± S.D. Please click here to view a larger version of this figure.
Figure 5: Simultaneous investigation of the transmigration of monocyte subpopulations under flow. (A) Time-lapse image sequences (from 0 to 20 min) of the recruitment of proangiogenic monocytes (CD16+) and non-angiogenic monocytes over time through TNFα-activated HUVEC. Scale bar = 40 µm; the apical and basal views are shown. The full sequences can be seen in Supplemental Movie 4 for apical and Supplemental Movie 5 for basal views. (B) Quantitation of the transmigration of human proangiogenic monocytes (HPMo: CD16+) and human non-angiogenic monocytes (HNMo) through a TNFα-activated HUVEC monolayer. N = 4 biological replicates, data are presented as mean ± S.D. *p < 0.05; Mann-Whitney test. (C) Time-lapse image sequences (from 0 to 20 min) of the recruitment of proangiogenic and non-angiogenic monocytes overtime through TNFα+VEGFA-activated HUVEC. Scale bar = 40 µm; the apical and basal views are shown. The full sequences can be seen in Supplemental Movie 6 for apical and Supplemental Movie 7 for basal views. (D) Quantitation of the transmigration of human proangiogenic monocytes (HPMo: CD16+) and human non-angiogenic monocytes (HNMo: CD16-) through TNFα+VEGFA-activated HUVEC monolayer. N = 4 biological replicates, data are presented as mean ± S.D. *p < 0.05; Mann-Whitney test. (E) Localization of CD16+ monocytes before (10 min) and after (15 min) transmigration through TNFα+VEGFA-stimulated HUVEC. The orthogonal views are shown. Scale bar = 40 µm Please click here to view a larger version of this figure.
Figure 6: Simultaneous investigation of the transmigration of CD14+ versus CD14- PBMC under flow. (A) Adhesion of CD14+versus CD14– PBMC to TNFα-activated HUVEC under flow. (B) Adhesion of CD14+versus CD14– PBMC to TNFα+VEGFA-activated HUVEC under flow. (C) Transmigration rate (%) of CD14+versus CD14– PBMC through TNFα-activated HUVEC under flow. (D) Transmigration rate (%) of CD14+versus CD14– PBMC through TNFα+VEGFA-activated HUVEC under flow. Data are mean ± S.D. N = 4 biological replicates. *p < 0.05; Mann-Whitney test. Please click here to view a larger version of this figure.
Supplemental Movie 1: View at the apical plane of pan-monocyte recruitment under flow. Expanded view of the recruitment of pan monocyte under flow at the apical plane. HUVEC were stained with CMFDA and the nuclei of monocytes were live-stained with Hoechst 33342. Scale bar = 50 µm Please click here to download this file.
Supplemental Movie 2: View at the basal plane of pan-monocyte recruitment under flow. Expanded view of the recruitment of pan monocyte under flow at the basal plane. HUVEC were stained with CMFDA and the nuclei of monocytes were live-stained with Hoechst 33342. Scale bar = 50 µm Please click here to download this file.
Supplemental Movie 3: Maximal projection of z-stacks of pan-monocyte recruitment under flow. Expanded view of the recruitment of pan monocyte under flow as shown in Supplemental Movies 1 and 2. HUVEC were stained with CMFDA and the nuclei of monocytes were live-stained with Hoechst 33342. Scale bar = 50 µm Please click here to download this file.
Supplemental Movie 4: View at the apical plane of the recruitment of monocyte subpopulations to TNFα-activated HUVEC. Expanded view at the apical plane of the simultaneous recruitment of monocyte subpopulations under flow to TNFα-activated HUVEC under flow. HUVEC were stained with CMFDA and the nuclei of monocytes were live-stained with Hoechst 33342. Human proangiogenic monocyte subpopulations were identified by the surface expression of CD16. Scale bar = 30 µm. Please click here to download this file.
Supplemental Movie 5: View at the basal plane of the recruitment of monocyte subpopulations to TNFα-activated HUVEC. Expanded view, at the basal plane, of the simultaneous recruitment of monocyte subpopulations under flow to TNFα-activated HUVEC under flow. HUVEC were stained with CMFDA and the nuclei of monocytes were live-stained with Hoechst 33342. The human proangiogenic monocyte (HPMo) subpopulation was identified by the surface expression of CD16. Scale bar = 30 µm. Please click here to download this file.
Supplemental Movie 6: View at the apical plane of the recruitment of monocyte subpopulations to TNFα+VEGFA-activated HUVEC. Expanded view, at the apical plane, of the simultaneous recruitment of monocyte subpopulations under flow to TNFα+VEGFA-activated HUVEC under flow. HUVEC were stained with CMFDA and the nuclei of monocytes were live-stained with Hoechst 33342. The human proangiogenic monocyte subpopulation was identified by the surface expression of CD16. Scale bar = 30 µm Please click here to download this file.
Supplemental Movie 7: View at the basal plane of the recruitment of monocyte subpopulations to TNFα+VEGFA-activated HUVEC. Expanded view, at the basal plane, of the simultaneous recruitment of monocyte subpopulations under flow to TNFα+VEGFA-activated HUVEC under flow. HUVEC were stained with CMFDA and the nuclei of monocytes were live-stained with Hoechst 33342. The human proangiogenic monocyte (HPMo) subpopulation was identified by the surface expression of CD16. Scale bar = 30 µm Please click here to download this file.
Here, we report a method detailing a study of how monocyte subpopulations transmigrate through the inflamed endothelial monolayer. The discussed method used confocal microscopy instead of phase-contrast microscopy, which is also used to study monocyte recruitment under flow3,11,19. One major advantage of using confocal microscopy for time-lapse imaging is the ability to unambiguously discriminate between transmigration and strong adhesion of monocytes. Though the phase-contrast microscopy-based method is also robust, it requires expertise in order to avoid mixing up transmigrated cells and strongly adherent cells. In this case, one needs to establish strict criteria for analysis in order to make a clear difference between these two states of the monocyte recruitment cascade. In addition, it is also important to perform an endpoint analysis by confocal microscopy in order to confirm the global trends observed by the phase-contrast microscopy. Thus, the direct use of confocal microscopy to investigate monocyte recruitment under flow provides clear results on the actual transmigration status of captured monocytes.
One of the major bottlenecks in executing leukocyte recruitment assays under flow and using a phase-contrast microscope is the time spent to perform the analysis and track individual cells from capture to transmigration through the cell-cell junction. Automation of such analysis is possible but difficult to perform due to phase-contrast similarities between crawling and transmigrated monocytes. Here we show by using confocal microscopy that monocyte transmigration was accompanied by a discontinuation of endothelial cell staining in the basal plane corresponding to the shape of the transmigrated monocytes underneath HUVEC. This positioning was confirmed by the orthogonal projection. The transition of monocyte localization occurred exclusively between cell-cell junctions indicative of a paracellular transmigration. This is consistent with our previous data, which showed that under flow in vitro, monocytes transmigrate exclusively through paracellular route with HUVEC3,18. To complement the method proposed here, it is possible to use non-blocking antibodies against junctional proteins such as VE-cadherin, JAMs, or PECAM1 in order to picture the potential sites of monocyte transmigration (paracellular vs. transcellular). We have confirmed that the black shapes surrounding the monocyte nuclei are a robust characteristic of transmigrated cells and a simple event that may be detectable by software. Even though a manual cell counting system is demonstrated here, the black shape formation around the leukocyte nucleus is a criterion that could be used to define leukocyte transmigration in automated analysis, thus saving a lot of time. We are currently working on developing an automated application for such analysis.
Fluorescence and confocal microscopy were previously used in the study of leukocyte recruitment. However, they were not used to investigate the recruitment of different subpopulations simultaneously. Here we propose a modality of using confocal microscopy to study the recruitment of leukocyte subtypes simultaneously in the same microenvironment. We show that confocal microscopy can be used to investigate simultaneously the migratory behaviors of different monocyte subpopulations. As an example, we have used CD16 expression to discriminate between proangiogenic and non-angiogenic monocytes in order to study the transmigration capacity of the two subpopulations in different inflammatory contexts. Consistent with our recent publication, by using the confocal microscopy modality, we have shown that the transmigration rate of CD16+ monocytes was lower when the endothelial cell monolayer was stimulated only by TNFα3. However, the combination of TNFα and VEGFA led to an increase in transmigration of proangiogenic monocytes. The transmigration rate was similarly high for non-angiogenic CD16– monocytes under both inflammatory conditions. We have previously shown that monocyte staining with the anti-CD16 antibody did not present any significant effect on transmigration, confirming this by the analysis of unlabeled monocytes after the transmigration assay, using confocal microscopy3. However, for new leukocyte subtypes or antibodies used to mark them, the labeling effect needs to be assessed. Using this method, up to three different populations of leukocytes can be simultaneously studied. This could be subpopulations that are functionally distinct or similar immune cell types. Although the focus here is on monocyte transmigration, other steps of their recruitment can also be analyzed by this method, including cell behavior before transmigration, such as capture, and migrational directionality. Post-transmigration events such as abluminal retention and reverse transmigration can also be investigated for different leukocyte populations, as an extension of this method. One limitation is the poor detection of the staining in the far-red channel in time-lapse imaging, as well as some overspill of the fluorescence signals that reduce the z-stack resolution. This was mainly related to the instrument used for confocal imaging. The use of image deconvolution could eventually help to improve the image quality and allow further analysis of the different steps of the leukocyte recruitment.
To study leukocyte recruitment under optimal conditions, it is important to check the activation status of the endothelial cell monolayer. Indeed, a deficient activation of endothelial cells leads to a global reduction in monocyte adhesion and transmigration. Endothelial cell activation can be checked by analyzing the expression level of adhesion molecules on endothelial cell surface such as ICAM1 and VCAM1. The level of these adhesion molecules must be increased compared to unstimulated endothelial cells. If no change is detectable in these endothelial adhesion molecules, the cultured HUVEC can be considered as not activated. Assessing the expression level of adhesion molecules can constitute a good quantitative control between different experiments using the same batch of HUVEC. However, the expression level of these adhesion molecules can also vary between different primary culture of endothelial cells limiting the consideration of a global threshold of ICAM1 or VCAM1. The change in shape of macrovascular endothelial cells such as HUVEC is also a good indicator of their activation. This latter change in phenotype allows a quick and qualitative assessment of HUVEC activation. However, the analysis of adhesion molecules might be a better choice for microvascular cells that don't show major shape-change upon activation with inflammatory cytokines.
For mechanistic studies, relevant negative controls of monocyte transmigration can be performed by using antibodies against endothelial adhesion molecules such as ICAM1, VCAM1 or on leukocyte surface such as Leukocyte Function-associated Antigen (LFA)-1. The use of a relevant negative control is essential for such mechanistic study in monocytes as they express Fc-receptors on their cell surfaces. The purity of monocytes after isolation is also important, in order to avoid contamination by other leukocyte populations and an underestimation of the rate of monocyte transmigration. Another critical parameter is the temperature, which needs to be set at 37 °C for all the assays in order to ensure all experimental observations are relevant and translate accordingly to human in vivo cell trafficking.
The authors have nothing to disclose.
We thank Dr. Paul Bradfield for manuscript reading and feedbacks. A. S. received financial support from the Sir Jules Thorn Charitable Overseas Trust Reg.,
Tissue Culture Flasks 75 cm2 | TPP | 90076 | Routine culture of isolated HUVEC |
µ-Slide VI 0.4 | IBIDI | 80606 | |
Centrifuge Tubes 15 mL | TPP | 191015 | |
Centrifuge Tubes 50 mL | TPP | 191050 | |
Collagen G | Biochrom | L 7213 | For coating of cell culture flasks |
Gelatin | Sigma-Aldrich | 1393 | For coating of cell culture flasks |
Dulbecco’s Phosphate Buffered Saline (without MgCl2 and CaCl2) | Sigma-Aldrich | D8537 | |
Dulbecco’s Phosphate Buffered Saline (with MgCl2 and CaCl2) | Sigma-Aldrich | D8662 | |
RPMI-1640 Medium | Sigma-Aldrich | R8758 | |
3-Way Stopcocks | BIO-RAD | 7328103 | |
penicillin 10000 u/ml streptomycine 10000 ug/ml fungizone 25 ug/ml | AMIMED | 4-02F00-H | |
Collagenase type 1 | Worthington | LS004216 | |
Medium 199 1X avec Earle's salts, L-Glutamine, 25 mM Hepes | GIBCO | 22340020 | |
Bovine Albumin Fraction V | ThermoFisher | 15260037 | |
Endothelial Cell Growth Supplement, 150mg | Millipore | 02-102 | |
Heparin Sodium | Sigma-Aldrich | H3149RT | |
Hydrocortisone | Sigma-Aldrich | H6909 | |
L-Ascorbic acid | Sigma-Aldrich | A 4544 | |
EDTA disodium salt dihydrate C10H14N2Na2O8 · 2H2O | APPLICHEM | A2937.0500 | |
CD144 (VE-Cadherin), human recombinant clone: REA199, FITC | Miltenyi Biotech | 130-100-713 | AB_2655150 |
CD31-PE antibody, human recombinant clone: REA730, PE | Miltenyi Biotech | 130-110-807 | AB_2657280 |
Anti-Podoplanin-APC, human recombinantclone: REA446, APC | Miltenyi Biotech | 130-107-016 | AB_2653263 |
BD Accuri C6 Plus | BD Bioscience | ||
µ-Slide I Luer | IBIDI | 80176 | |
CMFDA (5-chloromethylfluorescein diacetate) | ThermoFisher | C2925 | |
Recombinant human TNFα | Peprotech | 300-01A | |
Recombinant human VEGFA | Peprotech | 100-20 | |
NE-1000 Programmable Syringe Pump | KF Technology | NE-1000 | |
Ficoll Paque Plus | GE Healthcare | 17-1440-02 | |
Anti-human CD14-PE, human recombinant clone: REA599, PE | Miltenyi Biotech | 130-110-519 | AB_2655051 |
Pan Monocyte Isolation Kit, human | Miltenyi Biotech | 130-096-537 | |
Anti-human CD16-PE, human recombinant clone: REA423, PE | Miltenyi Biotech | 130-106-762 | AB_2655403 |
LS columns | Miltenyi Biotech | 130-042-401 | |
QuadroMACS Separator | Miltenyi Biotech | 130-090-976 | |
Hoechst 33342, Trihydrochloride, Trihydrate | ThermoFisher | H1399 | |
Silicone tubing | IBIDI | 10841 | |
Elbow Luer Connector | IBIDI | 10802 | |
Female Luer Lock Coupler | IBIDI | 10823 | |
Luer Lock Connector Female | IBIDI | 10825 | |
In-line Luer Injection Port | IBIDI | 10820 | |
Ar1 confocal microscope | Nikon | ||
40X objective | Nikon | 40x 0.6 CFI ELWD S Plane Fluor WD:3.6-2.8mm correction 0-2mm | |
ImageJ Software | NIH |