Matrigel (BD Biosciences, USA) was diluted at a ratio of 1:8, and 35 µL was added to the Transwell upper chamber (8 µm, Corning, USA). The Transwell chambers were placed in a 24‑well plate and incubated overnight at 4 °C. Briefly, 200 μL (HTR‑8/SVneo, 1 × 105 cells/well) DMEM/F‑12 suspension without 10% FBS was added to the upper chamber, and 600 μL DMEM/F‑12 containing 10% FBS was added to the lower chamber. According to different experimental requirements, cells pretreated with rh-CSF1(2 ng/mL, R&D Systems, USA) or vehicle were added in the upper chamber. The cells were cultured for 48 h at 37 °C in a 5% CO2 incubator. The 24‑well plate was removed, and the upper chamber medium and nonpenetrating cells were gently wiped off with a cotton swab, washed three times with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, and stained with crystal violet for 20 min. Thereafter, random photographs were acquired under an inverted microscope (× 200), and 5 visual fields were counted in each chamber. The number of invaded cells was counted using ImageJ software (National Institute of Mental Health, USA).
Rh csf1
Manufactured by R&D Systems
Sourced in United States
Rh-CSF1 is a recombinant human colony-stimulating factor 1 (CSF1) protein. CSF1 is a hematopoietic growth factor that regulates the production, differentiation, and function of macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (1 mentions)
Upper transwell chamber, by Corning (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Restore plus western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Apoptosis array kit, by R&D Systems (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
Rhrankl, by R&D Systems (1 mentions)
2 protocols using rh csf1
1
Cell Invasion Assay using Matrigel
2
Inhibitory Effects of mAb 3.1 on Feline Osteoclasts
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To further analyze the inhibitory properties of mAb 3.1, feline osteoclasts were targeted with the antibody. Feline bone marrow cells were differentiated for 10 days in rhRANKL (30 ng/ml, R&D Systems) and rhCSF-1 (10 ng/ml, R&D Systems) in low adherence tissue culture plates (Corning) (kindly provided by Seungmee Lee (Roslin Institute)). Anti-CSF-1R mAb 3.1 hybridoma supernatant or an irrelevant anti-CCR2 hybridoma supernatant was added to the cells at 1/6 of the total culture volume. One control well received no CSF-1 after the initial differentiation of the cells. The culture medium and supplements were renewed at 48 h. Cells were lysed on day 4 using 1 ml/well of lysis buffer 15 (R&D Systems, Apoptosis Array Kit) for 30 min at 4°C. The protein mixture was assessed by western blotting. Membranes were probed with anti-pAkt (Ser 473) and total Akt (both Cell Signaling). Antibodies were used sequentially on the same membrane, after stripping the nitrocellulose membrane using Restore PLUS Western Blot Stripping Buffer (Thermo Scientific). Secondary antibody was swine anti-rabbit HRP-conjugated.
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