Anti cd44 antibody

Avidin antibody conjugate was synthesized using EasyLink Avidin Conjugation kit (Abcam) following the manufacturer’s instructions. Briefly, 200 µg of avidin reagent was mixed with 200 μg of anti-CD44 antibody (Abcam, prod. No. ab119335). The antibody was mixed with PCL-chit-PEGb MPs (200 mg).

For the analysis of CD44 expression at a protein level following the treatment with HA at different concentrations (HA 1, HA 0.5, and HA 0.2) for 3 and 7 days, 1 × 10⁶ MSC were lysed using RIPA buffer (Thermo) mixed with halt protease inhibitor cocktail (Thermo). Untreated cells acted as control. Protein concentration was determined by Bradford assay (Bio-Rad) using BSA at known concentration (1 µg/µl) to build the standard curve. Forty μg of whole cell extract were loaded onto 12% acrylamide SDS-PAGE gel (Bio-Rad) and run for 40 minutes at 25 mA constant. Proteins were then transferred to a nitrocellulose membrane for Western blot analysis. Blots were incubated with primary anti-CD44 antibody (Abcam, 1:2000) and anti-GAPDH (Abcam, 1:20,000) overnight. Then, horseradish peroxidase (HRP)-conjugated secondary antibodies were used to incubate the membrane for 1 hr. Bands were visualized using a SuperSignal West Dura Chemiluminescent Substrate (Thermo) and images were acquired with ChemiDoc XRS + System and Image Lab software (Bio-Rad). Densitometry analysis was performed using ImageJ software (NIH).

Hoechst33342 (H3570) was purchased from Invitrogen (Carlsbad, CA, USA). Gemcitabine (G0367) was provided by the Tokyo Chemical Industry (Tokyo, Japan). EGCG (E4143), catalase (C100), and superoxide dismutase (SOD) (S5395) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Trequinsin hydrochloride was obtained from Toronto Research Chemicals (Toronto, ON, Canada). EGF and FGF were purchased from BD Bioscience, (Franklin Lakes, NJ, USA). B27 were purchased from Invitrogen. Antibodies used for immunofluorescence analysis and immunoblotting consisted of an anti-FOXO3 antibody (Abcam, Cambridge, MA, USA, ab109629), anti-CV-Caspase-3 antibody (Cell Signaling Technology, #9661S), anti-β-actin antibody (Sigma-Aldrich, 061M4808), anti-CD44 antibody and anti-PDE3A (Abcam, ab112534) were used for Western blotting (Abcam, ab51037); Alexa Fluor 555-conjugated secondary antibody (Invitrogen, A21428) were used for immunofluorescence analysis. A FITC-labeled anti-CD44 antibody used in flow cytometory analysi was purchased from Miltenyi Biotec, (130-095-195 Bergisch Gladbach, Germany). Akt activity was evaluated by using assay kit obtain from Millipore (Billerica, MA, USA CBA019). Accumax was purchased from Innovative Cell Technologies (San Diego, CA, USA). The AST/ALT activity assay kit was purchased from Wako. EGCG analogs were synthesized as described previously^{35 (link)}.

Annexin V-APC/PI was used to analyze the apoptosis level of PANC-1 and 6606PDA cells treated with gemcitabine, MIT, or both for 48 h. Anti-CD44 antibody (Abcam) as a CSC marker was employed to mark PANC-1 cells. APC anti-mouse CD206 antibody (BioLegend) and anti-iNOS AF 488-conjugated antibody (Invitrogen) were used to stain macrophages to identify the type of macrophages. After treatment for 48 h, RAW264.7 cells were harvested and transferred to a 15 ml tube. Antibodies were added at a ratio of 1:500 and incubated at room temperature for 10 min in the dark. Then, 10,000 cells were analyzed.