Anti-CD147 antibody [MEM-M6/1]

• Flow Cyt

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Flow Cytometry - Anti-CD147 antibody [MEM-M6/1] (AB666)

Overlay histogram showing peripheral blood lymphocytes stained with ab666 (red line). The cells were incubated with the antibody (ab666, 1 μg/1x10⁶ cells) for 30 minutes at 4°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 minutes at 4°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2 μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed gating on peripheral blood lymphocytes.

• Flow Cyt

Unknown

Flow Cytometry - Anti-CD147 antibody [MEM-M6/1] (AB666)

Flow cytometry of human peripheral blood cells with ab666 at 1 µg/ml

• Flow Cyt

Lab

Flow Cytometry - Anti-CD147 antibody [MEM-M6/1] (AB666)

Flow cytometry overlay histogram showing wild-type A549 (green line) and BSG knockout A549 cells (ab273748) stained with ab666 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab666) (1x10⁶ in 100μl at 10 μg/ml) for 30 min at 4°C.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at 4°C.

Isotype control antibody was mouse IgG1κ (ab170190) used at the same concentration and conditions as the primary antibody (wild-type A549 - black line; BSG knockout A549 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD147 antibody [MEM-M6/1] (AB666)

IHC image of CD147 staining in human normal heart muscle formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab666, 5 μg/ml, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• WB

Lab

Western blot - Anti-CD147 antibody [MEM-M6/1] (AB666)

Lanes 1 - 4 : Merged signal (red and green). Green - ab666 observed at 55-70 kDa. Red - loading control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.

ab666 was shown to react with CD147 in wild-type A549 cells in western blot with loss of signal observed in BSG knockout cell line ab273748 (knockout cell lysate ab275500). Wild-type and BSG knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab666 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD147 antibody [MEM-M6/1] (ab666) at 1 µg/mL

Lane 1:

Wild-type A549 cell lysate at 30 µg

Lane 2:

BSG knockout A549 cell lysate at 30 µg

Lane 2:

Western blot - Human BSG (CD147) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-bsg-cd147-knockout-a549-cell-line-ab273748'>ab273748</a>)

Lane 3:

Raji cell lysate at 30 µg

Lane 4:

Jurkat cell lysate at 30 µg

Predicted band size: 42 kDa

Observed band size: 55-70 kDa

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• WB

CiteAb

Western blot - Anti-CD147 antibody [MEM-M6/1] (AB666)

CD147 western blot using anti-CD147 antibody [MEM-M6/1] ab666. Publication image and figure legend from Colangelo, N. W. & Azzam, E. I., 2020, Cell Commun Signal, PubMed 32033611.

ab666 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab666 please see the product overview.

Irradiation of glioblastoma cells increases the levels of CD147 protein in their EVs. a Immunoblots for CD147 on the EVs of T98G and U-87 MG cells are shown for the 0 or 8 Gy dose at 24, 48, and 72 h for three experiments. The ‘C’ label represents the 0 Gy control samples, and the ‘IR’ label is for samples receiving 8 Gy of ionizing radiation. Immunoblot for CD147 in lysates of T98G cells, harvested after the 72 h collection of EVs, was also performed. CD147 levels were normalized relative to the Ponceau S stain. Graphical representations of these results are also displayed. b Immunoblot for CD147 on EVs collected at 24 h from T98G cells receiving increasing doses of γ-rays. The level of CD147 in the cells at 24 h was also measured. c Immunoblot for CD147 in EVs from T98G cells with CD147 knockdown, showing that CD147 still increases in response to γ-irradiation, even in partial knockdowns. Plasmid 1 clone 1 was the knockdown used in experiments. The scramble control (Scr) has saturated signal due to the high levels of CD147 in its EVs relative to the knockdowns. Clones 2 and 3 of plasmid 1 and clones 1, 3, and 4 of plasmid 2 demonstrate that even following knockdown, CD147 is increased in EVs when cells are irradiated. Immunoblots for CD147 on the cell lysates of the selected clones is also shown relative to a control c to provide a relative knockdown efficiency for cellular CD147. d Immunoblots on EVs of 0 or 8 Gy γ-irradiated U-118 MG cells, with the EVs from γ-irradiated U-87 MG cells used as a positive control. Despite EVs from U-118 MG cells not having CD147, they contained MCT1 and MCT4, which are found in EVs and co-localize with CD147. Ponceau S Red Stain was used as loading control. e Sucrose gradients determined that CD147 and CD63 sediment together at the correct density for EVs

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