Spheres were formed in 2.5% (v/v) methylcellulose (M0512, Sigma) hanging droplets using 1,000 cells total in a 2:1 ratio PSC: cancer cell. Spheres were collected 24 hours later and suspended in organotypic mixture (10.5 volumes high concentration Collagen (354249, Corning, 2 mg/mL final concentration), 7 volumes Matrigel, 1 volume HEPES (1 M, pH 7.5, H7006, Sigma) and 21.5 volumes relevant cell culture medium, with 1 M NaOH added dropwise to neutralise the pH), before being seeded into wells of a 96 well plate. Culture medium containing relevant treatments was added on top of gels once set. Gels were imaged using an Axiovert 135 (Carl Zeiss MicroImaging LLC) camera and percentage invasive area quantified using ImageJ (National Institutes of Health), using the following equation: % invasive area = ((total area - central area)/central area) x 100. At the end of the protocol, spheroid gels were washed once in PBS, fixed in 4% PFA for 20 minutes and washed three times in PBS. Z-stack images of fluorescently labelled spheres were taken using a Zeiss LSM Confocal 880 microscope.
H7006
Manufactured by Merck Group
H7006 is a laboratory equipment product designed for general use in research and scientific applications. It serves as a basic tool for various laboratory procedures and experiments. The core function of this product is to provide a standard and reliable piece of equipment for conducting laboratory work. Further details on its specific intended use or features are not available.
Automatically generated - may contain errors
3 protocols using h7006
1
Spheroid Invasion Assay Protocol
2
3D Spheroid Invasion Assay
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Spheres were formed in 2.5% (v/v) methylcellulose (M0512, Sigma) hanging droplets using 1000 cells total in a 2:1 ratio PSC: cancer cell. Spheres were collected 24 h later and suspended in organotypic mixture (10.5 volumes high concentration Collagen (354249, Corning, 2 mg/mL final concentration), 7 volumes Matrigel, 1 volume HEPES (1 M, pH 7.5, H7006, Sigma) and 21.5 volumes relevant cell culture medium, with 1 M NaOH added dropwise to neutralise the pH), before being seeded into wells of a 96 well plate. Culture medium containing relevant treatments was added on top of gels once set. Gels were imaged using an Axiovert 135 (Carl Zeiss MicroImaging LLC) camera and percentage invasive area quantified using ImageJ (National Institutes of Health), using the following equation: % invasive area = ((total area − central area)/central area) × 100. At the end of the protocol, spheroid gels were washed once in PBS, fixed in 4% PFA for 20 min and washed three times in PBS. Z-stack images of fluorescently labelled spheres were taken using a Zeiss LSM Confocal 880 microscope.
3
Correlative Microscopy of LAMP1-GFP Cells
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CLEM of HeLa LAMP1-GFP cells infected by STM WT was performed as previously described5 (link). Briefly, HeLa LAMP1-GFP cells were grown on MatTek dishes with a gridded coverslip (MatTek P35G-1.5-14-CGRD-D) and infected with the respective STM strains at MOI 75. Cells were fixed with 2% paraformaldehyde (PFA, Sigma-Aldrich, 158127) and 0.2% glutaraldehyde (GA, Sigma-Aldrich 111-30-8) in 0.2 M HEPES (Sigma-Aldrich, H7006) for 30 min prior to LM. After rinsing the cells for three times with 0.2 M HEPES buffer, unreacted aldehydes were blocked by incubation with 50 mM glycine in buffer for 15 min, followed by rinses in buffer. CLSM was performed and ROIs were chosen. Afterwards cells were fixed with 2.5% glutaraldehyde and 5 mM CaCl2 in 0.2 M HEPES in preparation for TEM. Further steps including post-fixation, dehydration, sectioning and imaging in the TEM were conducted as previously described5 (link).
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