Journal: Scientific Reports

Article Title: A cell based assay for evaluating binding and uptake of an antibody using hepatic nonparenchymal cells

doi: 10.1038/s41598-021-87912-6

Figure Lengend Snippet: Cellular binding and internalization assay using mouse NPC. ( a ) Cellular dot plot after staining with CD146 and CD45. X axis (Pacific Blue) and Y axis (FITC) shows CD45 and CD146, respectively. CD146 high/CD45 low, CD146 low/CD45 low, and CD146 low/CD45 high population were named as P1, P2, and P3, respectively. ( b ) Histogram of the FcγRIIB expression in each population. X axis (APC) shows the fluorescence of Alexa Fluor 647. Red shows staining with anti- FcγRIIB antibody and blue shows unstaining. ( c ) Concentration-dependent binding of the antibody in P2. The antibody was incubated at 4 °C for 60 min, and fluorescence was detected by FCM. The data were fitted with the receptor-ligand binding equation. ( d ) Concentration-dependent internalization rate of the antibody in P2. The antibody was incubated at 37 °C for 5 min. The data were fitted with the Michaelis–Menten equation. Each point shows the mean ± SD (n = 3).

Article Snippet: The following materials were purchased from commercial sources: Clear Immuno 384-Well Plate (Thermo Scientific, 8755), an anti-human IgG antibody (Bethyl Laboratories, A80-319A), Streptavidin-PolyHRP80 (Stereospecific Detection Technologies, SP80D50), Bovine Serum Albumin (BSA) for ELISA (Sigma-Aldrich, A7030), TMB Substrate (Surmodics, TMBS-1000–01), Carbonate-Bicarbonate Buffer capsule (Sigma-Aldrich, C3041), 96-well cell culture plate (Costar, 3799), BSA for cell assay (Sigma-Aldrich, A9418), Fetal bovine serum (FBS) (Sciencell, 0500), anti-mouse CD45 antibody-VioBlue (Miltenyi, 130-110-664), anti-mouse CD146 antibody-FITC (Miltenyi, 130-102-230), anti-human CD31 antibody-Pacific Blue (BioLegend, 303114), anti-non human primate CD45 antibody-APC (Miltenyi, 130-091-900), anti-CD32B (FcγRIIB) antibody (Sino Biological, 90014-R046), Liver Perfusion Medium (Gibco, 17701-038), Endothelial cell medium (Sciencell, 1001), Hepatocyte Culture Medium (Lonza, CC-3198), Alexa Fluor 488 Labeling Kit (Invitrogen, A20181), Alexa Fluor 647 Labeling Kit (Invitrogen, A20173).

Techniques: Binding Assay, Staining, Expressing, Fluorescence, Concentration Assay, Incubation, Ligand Binding Assay

Journal: Scientific Reports

Article Title: A cell based assay for evaluating binding and uptake of an antibody using hepatic nonparenchymal cells

doi: 10.1038/s41598-021-87912-6

Figure Lengend Snippet: Cellular binding and internalization assay using monkey NPC. ( a ) Cellular dot plot after staining with CD31 and CD45. X axis (Pacific Blue) and Y axis (APC) shows CD31 and CD45, respectively. CD31 low/CD45 high, CD31 mid-low/CD45 mid-high, CD31 mid-low/CD45 high, CD31 high/CD45 high, CD31 mid-high/CD45 mid-low, and CD31 low/CD45 low population were named as P1, P2, P3, P4, P5, and P6, respectively. ( b ) Histogram of the FcγRIIB expression in each population. X axis (FITC) shows the fluorescence of Alexa Fluor 488. Red shows staining with anti- FcγRIIB antibody and blue shows unstaining. ( c ) Concentration-dependent binding of the antibody in P2 cells. The antibody was incubated at 4 °C for 60 min, and fluorescence was detected by FCM. The data were fitted with the receptor-ligand binding equation. ( d ) Concentration-dependent internalization rate of the antibody in P2 cells. The antibody was incubated at 37 °C for 10 min. The data were fitted with the Michaelis–Menten equation. Each point shows the mean ± SD (n = 3).

Article Snippet: The following materials were purchased from commercial sources: Clear Immuno 384-Well Plate (Thermo Scientific, 8755), an anti-human IgG antibody (Bethyl Laboratories, A80-319A), Streptavidin-PolyHRP80 (Stereospecific Detection Technologies, SP80D50), Bovine Serum Albumin (BSA) for ELISA (Sigma-Aldrich, A7030), TMB Substrate (Surmodics, TMBS-1000–01), Carbonate-Bicarbonate Buffer capsule (Sigma-Aldrich, C3041), 96-well cell culture plate (Costar, 3799), BSA for cell assay (Sigma-Aldrich, A9418), Fetal bovine serum (FBS) (Sciencell, 0500), anti-mouse CD45 antibody-VioBlue (Miltenyi, 130-110-664), anti-mouse CD146 antibody-FITC (Miltenyi, 130-102-230), anti-human CD31 antibody-Pacific Blue (BioLegend, 303114), anti-non human primate CD45 antibody-APC (Miltenyi, 130-091-900), anti-CD32B (FcγRIIB) antibody (Sino Biological, 90014-R046), Liver Perfusion Medium (Gibco, 17701-038), Endothelial cell medium (Sciencell, 1001), Hepatocyte Culture Medium (Lonza, CC-3198), Alexa Fluor 488 Labeling Kit (Invitrogen, A20181), Alexa Fluor 647 Labeling Kit (Invitrogen, A20173).

Techniques: Binding Assay, Staining, Expressing, Fluorescence, Concentration Assay, Incubation, Ligand Binding Assay

Journal: Purinergic Signalling

Article Title: Adenosine enhances cisplatin sensitivity in human ovarian cancer cells

doi: 10.1007/s11302-018-9622-7

Figure Lengend Snippet: Expression and functional activity of adenosine receptors in A2780, A2780CisR, and HEY cells. a Gene expression of adenosine receptors using RT-PCR. b Protein expression of adenosine receptors using Western blot. c Concentration-dependent effect of adenosine on cAMP-related luminescence in A2780 cells. d–f Concentration-dependent effect of SLV320, PSB603, and SCH 442416 (selective A1, A2B, and A2A antagonists, respectively) on 200 μM adenosine-induced luminescence in A2780 cells

Article Snippet: Rabbit anti-A1, A2A, A2B, and A3 adenosine receptor antibodies were obtained from Alomone labs (Israel).

Techniques: Expressing, Functional Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Concentration Assay