TE4 and TE4CCR7+ cells were labeled with green 5-chloromethylfluorescein diacetate (CMFDA; Invitrogen). To investigate the effect of epithelial cells on cell adhesion, normal human lung lymphatic microvascular endothelial cells (HMVEC-LLy; Lonza Walkersville Inc., Walkersville, MD) were used in this assay, and HMVEC-LLy was labeled with a red fluorescent dye (CMPTX; Invitrogen). Chambers were confluent with a monolayer of HMVEC-LLy. We seeded 5 × 104 cells/well TE4 or TE4CCR7+ and incubated for 10 min at 37°C with or without human recombinant CCL21/SLC (R&D Systems). A blocking assay was performed using mouse monoclonal anti-CCR7 antibody (10 μg/ml; R&D Systems), mouse monoclonal anti-intercellular adhesion molecule (ICAM)-1 (10 μg/ml; Abcam, Cambridge, England), or anti-ICAM-2 antibody (10 μg/ml; Abcam). The number of attached cells in five randomly-selected fields was semiautomatically counted using BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan). These procedures were repeated at least three times.
Cmptx
Manufactured by Thermo Fisher Scientific
The CMPTX is a laboratory instrument designed for conducting complex biomolecular analyses. It features advanced imaging and detection capabilities to support a wide range of scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Cmfda, by Thermo Fisher Scientific (1 mentions)
Biorevo bz 9000 microscope, by Keyence (1 mentions)
Hmvec lly, by Lonza (1 mentions)
Te 2000 confocal microscope, by Nikon (1 mentions)
Rat tail collagen 1, by BD (1 mentions)
Yo pro 1, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
A1r inverted confocal microscope, by Nikon (1 mentions)
Glass bottom multi well plates, by MatTek (1 mentions)
Lsrfortessa, by BD (1 mentions)
Celltracker cmfda, by Thermo Fisher Scientific (1 mentions)
Lsm 510 axiovert 200 m, by Zeiss (1 mentions)
5 protocols using cmptx
1
Adhesion of CCR7+ T cells to Lymphatic Endothelial Cells
2
Endpoint and Live-cell 3D Cell Migration Assays
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For endpoint cell migration assays, 12Z were mixed with DMEM +2.2 mg/ml rat tail collagen I (BD Biosciences) on ice, placed in a standard 96-well tissue culture plate at 50 μl and 5000 cells per well, spun for 5 min at 300 × g, and allowed to polymerize at 37o C. 50 μl full serum media containing inhibitors, growth factors (added 30 min following inhibitors when appropriate) or the relevant buffer control was then added to the wells, and the plate was incubated for 24 h. Gels were then fixed with 1% paraformaldehyde, stained with YO-PRO-1 (Invitrogen), and imaged with a Nikon A1R inverted confocal microscope. Migration experiments were interpreted using a modified spot finding algorithm64 (link)13 (link) in Matlab (Mathworks; Natick, MA). Live-cell 3D migration (Fig. S2 ) was assessed using data as previously described13 (link). Cells were labeled with a cell-tracker dye (CMPTX; Invitrogen), mixed at 5 × 105 cells/mL with DMEM +2.2 mg/mL pH-neutralized collagen-I (BD Biosciences) in glass-bottom multi-well plates (MatTek), polymerized for 30 min at 37 °C, and overlaid complete media overnight. Cells were stimulated 4 h prior to imaging on an environment-controlled Nikon TE2000 confocal microscope for 16 h. Bitplane Imaris software was used to track cells, and Matlab (Mathworks) was used to calculate the random motility coefficient.
3
Detecting Macrophage-Induced Invadopodia Formation
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The 405 gelatin-labeled Mattek dishes were prepared as previously described43 (link)59 (link). Tumor cells were plated in complete media for 6 h on the Alexa 405-labeled gelatin dishes. Dishes were fixed and immunostained for cortactin and Tks5 as previously described. Cells were imaged on a wide-field microscope (Inverted Olympus IX70) and images were acquired with a cooled CCD camera (Sensicam QE cooled CCD camera) with a 60 × NA = 1.4 oil immersion objective using IP Laboratory 4.0 software. Invadopodia were detected as punctate structures that were positive for both cortactin and tks5 and capable of degrading Alexa 405-gelatin.
To detect macrophage-induced invadopodia, MDA-MB-231 cells were serum-starved for 16 h. BAC1.2F5 cells were cell tracker-labeled (CMPTX, Invitrogen). A total of 25 K MDA-MB-231 cells were incubated with 125 K BAC1.2F5 cells in serum-starvation media on 405-labeled gelatin-coated dishes for 6 h, fixed and immunostained for invadopodium markers as described above. For MTLn3 experiments, tumor cells were serum starved for 4 hrs before being plated with BAC1.2F5 cells for 6 hrs as described above.
To detect macrophage-induced invadopodia, MDA-MB-231 cells were serum-starved for 16 h. BAC1.2F5 cells were cell tracker-labeled (CMPTX, Invitrogen). A total of 25 K MDA-MB-231 cells were incubated with 125 K BAC1.2F5 cells in serum-starvation media on 405-labeled gelatin-coated dishes for 6 h, fixed and immunostained for invadopodium markers as described above. For MTLn3 experiments, tumor cells were serum starved for 4 hrs before being plated with BAC1.2F5 cells for 6 hrs as described above.
4
Quantifying Phagocytic Engulfment Assay
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Phagocytosis was assessed as previously described (Cvetanovic and Ucker, 2004 (link)). Target cells were labeled green with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA; 0.2 μM; Molecular Probes, Eugene, OR) and washed extensively and then were induced to undergo apoptotic cell death, killed pathologically by heat treatment, or left untreated. Responder cells were labeled red with the tracking dye CMPTX (2 μM; Molecular Probes), washed extensively, and incubated overnight before coculture with apoptotic target cells. Responder cells then were washed with PBS, followed by a wash with PBS supplemented with 0.4 mM Na2EDTA, followed by a wash with 0.05% trypsin-EDTA (Mediatech) to remove any bound targets. Cells then were gently lifted by scraping, and analyzed cytofluorimetrically on the BD LSR Fortessa (BD Biosciences, Franklin Lakes, NJ). Bound targets that that had not been engulfed did not remain adherent during these procedures. Cells that stained positively for CMPTX (Exλ = 577 nm; Emλ = 602 ± 15 nm) and also were CFDA positive (Exλ = 488 nm; Emλ = 530 ± 15 nm) represented responders that had engulfed targets. Phagocytosis is represented as the fraction of responder cells that are CFDA+.
5
Interaction of Endothelial Cells and Macrophages
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Endothelial spheroids consisting of 400 cells were prepared as previously described (Michaelis et al., 2013) and cultured with 200 μl EBM medium. MACs were infected with B. henselae at increasing MOIs (200, 250, 300) or left uninfected for 24 h prior to spheroid formation. For HUVEC-MAC co-culture spheroids, MACs and HUVECs were mixed at a ratio of 1:3 before spheroid formation. Alternatively, MACs (1.75 × 10 5 cells per well infected or uninfected) were mixed with the spheroid collagen gel or seeded onto the spheroid gels with growth medium. Lastly, spheroid gels were cultured with 400 μl conditioned EBM medium, collected from MACs infected for 24 h prior to spheroid formation. The rate of endothelial sprouting was assessed after 12 h by computer-assisted inverse light microscopy. For red-green co-culture spheroids, HUVECs and MACs were first stained with red (CMPTX) and green (CMFDA) cell tracker (Molecular Probes, Life Technologies) respectively. A total of 30 μl spheroid collagen gel suspension and 30 μl EBM was added to each well of an angiogenesis micro-slide (ibidi). The localization of MACs and HUVECs within the spheroids was analysed using CLSM (LSM 510 Axiovert 200M, Zeiss). At least 10 spheroids were analysed for each condition.
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