Sodium alginate (~44% G content) and collagen (PureCol® EZ Gel) were purchased from Sigma Aldrich (UK). The OX1-19 iPSC line was generated and provided by S. Cowley at the University of Oxford (UK) [38 ]. Neurobasal media, N2 and B27 supplements were purchased from Life Technologies™ (UK). Primary and secondary antibodies for immunofluorescence microscopy were purchased from Abcam (UK). RNA isolation, cDNA synthesis and primer kits for RT-PCR were purchased from Qiagen, Bio-Rad and Primerdesign (all UK), respectively. All other reagents were purchased from Sigma Aldrich (UK) and used without further modification.
Purecol ez gel
Manufactured by Merck Group
Sourced in United States
PureCol® EZ Gel is a laboratory product manufactured by the Merck Group. It is a collagen-based solution designed for various research and cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Tissue train plate, by Flexcell (2 mentions)
Dasatinib, by Cell Signaling Technology (2 mentions)
Eclipse ti, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Ab92516, by Abcam (1 mentions)
Gb22303, by Wuhan Servicebio Technology (1 mentions)
Tcs sp8, by Leica (1 mentions)
Lipoprotein deficient serum from fetal calf, by Merck Group (1 mentions)
7 protocols using purecol ez gel
1
Alginate-Collagen hydrogel for iPSC culture
2
Translocation of Calreticulin in Tumor Cells
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Tumor cells (5×105 cells per well) were planted into a 24-well plate precoated with gel (PureCol® EZ Gel, Sigma, 1:150) and treated with BI2536 for 24 h. To analyze the translocation of CRT in immunofluorescence, the samples were fixed in 4% paraformaldehyde (PFA) before permeabilization with 0.1% TritonX-100, and then stained with a rabbit anti-mouse anti-CRT antibody (ab92516, Abcam, 1:100) for 30 min. Subsequently, the cells were stained with a secondary antibody FITC goat anti-rabbit IgG (GB22303, Servicebio, 1:200). The cytoskeleton was incubated with phalloidin (C2207S, Beyotime, 1:1000) and cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime). The fluorescence information was acquired by laser confocal microscopy (NIKON Eclipse Ti). For flow cytometry, treated tumor cells were incubated with anti-CRT and secondary FITC goat anti-rabbit IgG as previously described. Then the samples were prepared into 100 μL of PBS suspension and subjected to FACScalibur detection.
3
Engineered Engineered Tissue Constructs
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BATs were prepared as previously described10 (link). RGD (Abcam, USA, #ab142698) and RGDS (Tocris Bioscience, USA, # 3498) were added to the neutralized mixture of Purecol EZ Gel (Sigma-Aldrich, USA, # 5074), 5 × low glucose DMEM, fetal bovine serum and C3H10T1/2 cells before the mixture was pipetted into each well of an untreated Tissue Train plate (Flexcell International Corp., USA, #TT-5001U). The gels were set in an incubator for two hours and BATs were covered with 2 ml complete media including RGD or RGDS.
4
Assembling Endometrial Organoids
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At passage 2, EnSC and gland-like organoid pellets were mixed at a ratio of 1:1 (v/v) and ice-cold PureCol EZ Gel (Sigma-Aldrich) added at a ratio of 1:20 (cell pellet: hydrogel). Samples were kept on ice until plating. The suspension was aliquoted in 20 µl volumes using ice-cold pipette tips into a 48-well plate, one droplet per well, and allowed to cure in the cell culture incubator for 45 min. Expansion medium supplemented with 10 nM E2 was overlaid and the medium was refreshed every 48 hr. For decidualization experiments, assembloid cultures were grown in expansion medium supplemented with E2 for 4 days to allow for growth and expansion. Assembloids were then either harvested or decidualized using different media as tabulated in Supplementary file 1 : Table 1 for a further 4 days. Again, the medium was refreshed every 48 hr and spent medium stored for further analysis. For tyrosine kinase inhibition, MDM was supplemented with 250 nM dasatinib (Cell Signaling Technology, Leiden, NL).
5
BMDM Seek Behavior in 3D Collagen Gel
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For analyzing BMDM seek behavior, 0.5 × 106 BMDMs stimulated for 24 hr with 20 ng/ml GM-CSF or 50 ng/ml M-CSF from EGFP-ubiquitin mice were re-suspended in 100 μL of cRPMI and mixed with 200 μL PureCol EZ Gel (5074, Sigma-Aldrich). The collagen cell mix was then applied to a three-sided chamber on a microscopy slide, created by sealing with paraffin wax and covering with a glass slip. The microscopy slide was then incubated upright in a 5% CO2, 37°C incubator for 1 h to allow the collagen gel to set. 250 μL of cRPMI was then layered on top of the collagen gel inside the chamber, the remaining side sealed with paraffin wax and slide taken immediately to a TCS SP8 (Leica) microscope containing a 37°C pre-heated imaging chamber for imaging of BMDM seek behavior over the space of 1 h. Seek behavior videos were processed and quantified using Imaris software.
6
Biaxial Tendon Tissue Engineering
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BATs were prepared as previously described [21 (link)] with some modifications. Human tendon cells were seeded in the mixture of Purecol EZ Gel (Sigma-Aldrich, USA, # 5074), 5× DMEM, and 5% Lipoprotein Deficient Serum from fetal calf (Sigma, USA, #S5394) with or without 50 µg/ml oxLDL and 5 ng/ml TGF-β. The cells were suspended in the media and other components. Then they were mixed with the neutralized Purecol EZ Gel. The mixture was pipetted between the anchor stems of untreated Tissue Train plate (Flexcell International Corp., USA, #TT-5001U) in each well. After setting the BATs in the incubator, 3ml 1x DMEM media with or without oxLDL and TGF-β was added to each well and the plate was incubated for 72 h (3 days) as the changes in collagen gel by the seeded cells are normally evident within 3 days [22 (link), 23 (link)]. The BATs were then scanned using an image scanner and the images were analyzed in ImageJ to measure the diameter of the BATs.
7
Endometrial Assembloid Culture and Decidualization
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At passage 2, EnSC and gland organoid pellets were mixed at a ratio of 1:1 (v/v) and icecold PureCol® EZ Gel (Sigma Aldrich) added at a ratio of 1:20 (cell pellet: hydrogel).
Samples were kept on ice until plating. The suspension was aliquoted in 20 µl volumes using ice-cold pipette tips into a 48-well plate, one droplet per well, and allowed to cure in the cell culture incubator for 45 minutes. Expansion medium supplemented with 10 nM E2 was overlaid and the medium was refreshed every 48 hours. For decidualization experiments, assembloid cultures were grown in expansion medium supplemented with E2 for 4 days to allow for growth and expansion. Assembloids were then either harvested or decidualized using different media as tabulated in Supplementary Table1 for a further 4 days. Again, the medium was refreshed every 48 hours and spent medium stored for further analysis. For tyrosine kinase inhibition, MDM was supplemented with 250 nM dasatinib (Cell Signaling Technology, Leiden, NL).
Samples were kept on ice until plating. The suspension was aliquoted in 20 µl volumes using ice-cold pipette tips into a 48-well plate, one droplet per well, and allowed to cure in the cell culture incubator for 45 minutes. Expansion medium supplemented with 10 nM E2 was overlaid and the medium was refreshed every 48 hours. For decidualization experiments, assembloid cultures were grown in expansion medium supplemented with E2 for 4 days to allow for growth and expansion. Assembloids were then either harvested or decidualized using different media as tabulated in Supplementary Table1 for a further 4 days. Again, the medium was refreshed every 48 hours and spent medium stored for further analysis. For tyrosine kinase inhibition, MDM was supplemented with 250 nM dasatinib (Cell Signaling Technology, Leiden, NL).
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