First, 1×106 A549 cells were incubated with mouse α-integrin α2β1 antibody (ab24697, Abcam) on ice for 30 min, followed by incubation with FITC goat α-mouse antibody (554001, BD) on ice for another 30 min. The samples were then washed three times and re-suspended in phosphate-buffered saline (PBS). The stained samples were analyzed using a BD FACSCanto II, and data were collected from 1×104 viable cells. Experimental data were analyzed with Flowjo7.2.2 software. For FACS sorting of cells with high expression levels of integrin α2β1, only the top 25% of the integrin α2β1-overexpressing cancer cells were collected and expanded for second-round selection. The subline from the first round of selection was designated A549+, and the subline from the second round of selection was designated A549++. The proliferation of A549, A549+ and A549++ cells was validated with a CCK8 assay, which indicated no significant difference among these cell lines (p = 0.1; Figure S1 ).
Ab24697
Manufactured by Abcam
Ab24697 is a lab equipment product offered by Abcam. It is a device used for experimental purposes in a laboratory setting. The core function of this product is to facilitate specific tasks or procedures required in a research or scientific environment.
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Lab products found in correlation
Facscanto 2, by BD (1 mentions)
Ab78614, by Abcam (1 mentions)
Mouse monoclonal anti ha, by Fortrea (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Cytarabine, by Merck Group (1 mentions)
Penicillin, by Biosera (1 mentions)
Streptomycin, by Biosera (1 mentions)
L glutamine, by Biosera (1 mentions)
Gentamycin, by Biosera (1 mentions)
Sodium pyruvate, by Biosera (1 mentions)
Amphotericin b, by Biosera (1 mentions)
Ab201336, by Abcam (1 mentions)
Mab13501, by R&D Systems (1 mentions)
Ve cadherin, by Santa Cruz Biotechnology (1 mentions)
Ep1607ihcy, by Abcam (1 mentions)
5 protocols using ab24697
1
Enrichment and Characterization of Integrin α2β1-Expressing Cancer Cells
2
Functional Inhibition Assay Using Integrin Antibodies
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The functional inhibition assay was performed by the antibody blockade method. During the pH adjustment process in the cell detachment assay, 5 μg/mL of specific neutralizing antibodies were added into the culture medium and incubated for an indicated period. The neutralizing antibodies used were as follows: anti-α2β1 integrin (ab24697; Abcam), anti-α3 integrin (MAB1952Z; Merk Millipore), anti-α5 integrin (ab78614; Abcam), anti-β2 integrin (MAB1962; Merk Millipore), and anti-β3 integrin (MAB1957; Merk Millipore).
3
Syndecan Signaling in Breast Cancer
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Dulbecco’s modified eagles medium (DMEM), fetal bovine serum (FBS), sodium pyruvate, L-glutamine, penicillin, streptomycin, amphotericin B and gentamycin were all obtained from Biosera LTD (Courtabo euf Cedex, France). The cytostatic agent cytarabine, E2 the of EGFR inhibitor AG1478, the AG1024 inhibitor of IGF-IR, and EGF were also purchased from Sigma Chemical Co. (St Louis, MO, USA). IGF was purchased from R&D systems (Minneapolis, USA). All other chemicals used were of the best commercially available grade. Antibodies used were mouse monoclonal anti-syndecan-4 (5G9; Santa Cruz), mouse monoclonal anti-syndecan-1 (B-A38; Abcam), mouse monoclonal anti-HA (Clone HA.11; Covance), rabbit polyclonal against IGF-IRβ (D23H3; Cell signaling), rabbit polyclonal against ERα (HC-20, sc-534; Santa Cruz), rabbit polyclonal against ERβ (H-150, sc-8974; Santa Cruz) and mouse monoclonal to integrin alpha 2 + beta1 [P1E6] (ab24697; Abcam).
4
Integrin-mediated Collagen Deposition
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We investigated the contribution of an integrin-mediated mechanism by adding 5 μg/ml α2β1 blocking antibody (AB24697, Abcam) to every medium change starting from the first passage following 12 days of culture. Control groups were maintained in media supplemented with an equal volume of PBS. The distribution of cells expressing collagen was determined by immunohistochemistry. At +5d, hydrogels were fixed in phosphate-buffered formalin for 24 hours, after which point they were soaked in O.C.T. overnight. They were then cryosectioned at 5 μm thickness. Sections were stained for Picrosirius Red, H&E, and immunohistochemistry (IHC) using a primary antibody against type 1 collagen in conjugation with a mouse-specific HRP/DAB detection kit as described above.
5
Xeno-Free Culture and Marker Analysis
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ECs, FBs, PCs, and KCs cultured under xeno‐free conditions were analyzed for surface and intracellular markers expression by flow cytometry and immunofluorescence microscopy using antibodies against: CD31 (WM59; Biolegend), CD45 (2D1; Biolegend), ZO‐1 (sc‐33725; Santa Cruz), VE‐cadherin (sc‐6458; Santa Cruz), vWF (ab201336; abcam) claudin‐5 (34‐1600; Invitrogen), PDGFR‐α (16A1; Biolegend), PDGFR‐β (18A2; Biolegend), CD90 (5E10; Biolegend), NG2 (9.2.27; Invitrogen), a‐SMA (1A4; Invitrogen), FAP (AF3715; Novus Biologics), integrins α2β1 (ab24697; abcam), α5β1 (NBP2‐52680; Novus Biologics), αvβ3 (sc‐7312; Santa Cruz), α6 (MAB13501; R&D systems), α3 (MAB1345; R&D systems), β4 (NBP1‐43369), CK14 (LL002; Novus Biologics), CK10 (EP1607IHCY, abcam), and occludin.
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