Tissue culture plates (48-well, Corning) were coated with purified human collagen type I (Sigma-Aldrich, #CC050), type III (Sigma-Aldrich, #CC054), type IV (Sigma-Aldrich, #C6745) or recombinant human versican (RayBiotech, #230-00833-50) at a concentration of 50 μg/ml, for 1 h (37 °C, 5% CO2) in line with previously published methods from our lab21 (link). KRT5+ cells from healthy controls and IPF patients were seeded (2.5 ×103) into each well and after 30 min to adhere, were imaged with a 4x objective on the JuLITM Stage Real-Time Cell History Recorder every 15 min for 48 h. The JPEG images generated were imported to Nikon NIS Elements v.4.50 for analysis.
Cc050
Manufactured by Merck Group
Sourced in United States
The CC050 is a laboratory equipment product from Merck Group. It is designed to perform core functions related to sample analysis and research. The detailed specifications and intended use of this product are not available for an unbiased and factual description.
Automatically generated - may contain errors
Lab products found in correlation
Af1086, by R&D Systems (2 mentions)
Erms da470, by Merck Group (2 mentions)
Af164, by R&D Systems (2 mentions)
Recombinant human gm csf, by Gentaur (2 mentions)
Maxisorp plate, by Thermo Fisher Scientific (2 mentions)
Cc054, by Merck Group (1 mentions)
Nis elements v4, by Nikon (1 mentions)
R4130, by Merck Group (1 mentions)
A6013, by Merck Group (1 mentions)
5 protocols using cc050
1
Collagen and Versican Modulate Lung Cell Migration
2
3D Tumor Spheroid Invasion Assay
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We seeded 1000 cancer cells/spheroids into a 35 or 81-well agarose tube (A6013, Sigma-Aldrich, USA), which was generated using a 3D Petri dish, a spheroid formation device developed by Microtissues (Microtissues® Inc., RI, USA). After seeding the cells for 1 min, add 1 mL of cell culture medium for a 35-well plate or 2 mL for an 81-well plate. Incubate at 37 °C with 5 % CO2 to promote the formation of tumor spheres. Macrophages and cancer cells were added to the cancer spheroids in a ratio of 5:1. They were then co-cultured in RPMI (R4130, Sigma-Aldrich, USA) supplemented with 10 % fetal bovine serum and 100 units/mL of penicillin/streptomycin. After a 2-day co-culturing period, the invasion into type I collagen (CC050, Sigma-Aldrich, USA) was initiated.
To begin, neutralize the collagen to achieve a pH value of 7.0–8.0. Then, embed the co-culture into 35-well agarose gel tubes. Following a 4-minute incubation, invert the agarose tube containing collagen embedding co-culture and continue incubating for 1 hour. Next, invert the tube and add RPMI medium containing 5 % FBS and 1 % Pen/Strep. Perform invasion assays for 2 days and acquire images using an inverted microscope [56] (link).
To begin, neutralize the collagen to achieve a pH value of 7.0–8.0. Then, embed the co-culture into 35-well agarose gel tubes. Following a 4-minute incubation, invert the agarose tube containing collagen embedding co-culture and continue incubating for 1 hour. Next, invert the tube and add RPMI medium containing 5 % FBS and 1 % Pen/Strep. Perform invasion assays for 2 days and acquire images using an inverted microscope [56] (link).
3
Quantitative Antibody Binding ELISA
4
Quantitative Antibody Binding ELISA
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Total IgGs were quantified using 96-well MaxiSorp plates (Nunc) coated with goat
anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470
(ERMs-DA470, Sigma-Aldrich) as a standard. To test specific binding of antibody
constructs, ELISA plates were coated with 2 μg ml−1 of type I
recombinant human collagen (Millipore, CC050), 2 μg ml−1 of an
anti-human LAIR1 antibody (clone DX26, BD Biosciences 550810), 1 μg
ml−1 of recombinant human GM-CSF (Gentaur), 2 μg
ml−1 of an anti-PD1 or an anti-SLAM antibody (R&D Systems,
AF1086 and AF164). Plates were blocked with 1% bovine serum albumin (BSA) and incubated
with titrated antibodies, followed by AP-conjugated goat anti-human IgG, Fcγ
fragment specific (Jackson Immuno Research, 109-056-098). Plates were then washed,
substrate (p-NPP, Sigma) was added and plates were read at 405 nm.
anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470
(ERMs-DA470, Sigma-Aldrich) as a standard. To test specific binding of antibody
constructs, ELISA plates were coated with 2 μg ml−1 of type I
recombinant human collagen (Millipore, CC050), 2 μg ml−1 of an
anti-human LAIR1 antibody (clone DX26, BD Biosciences 550810), 1 μg
ml−1 of recombinant human GM-CSF (Gentaur), 2 μg
ml−1 of an anti-PD1 or an anti-SLAM antibody (R&D Systems,
AF1086 and AF164). Plates were blocked with 1% bovine serum albumin (BSA) and incubated
with titrated antibodies, followed by AP-conjugated goat anti-human IgG, Fcγ
fragment specific (Jackson Immuno Research, 109-056-098). Plates were then washed,
substrate (p-NPP, Sigma) was added and plates were read at 405 nm.
5
Cell Attachment Assay on Collagen I
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Cell attachment assay was performed based on a published protocol [14] . Briefly, HeLa cell were detached by non-enzymatic dissociation buffer (13151014, Thermosci Fisher) and reseeded onto 96-well polystyrene plate coated with 40 µg/ml collagen I (CC050, Millipore) for 30 mins. After the non-adherent cells were washed away by DMEM media, the remaining attached cells were quantified by crystal violetbased spectrophotometric method as previously described [2] .
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