Anti-CD86 antibody [EPR21962]

• Flow Cyt

Lab

Flow Cytometry - Anti-CD86 antibody [EPR21962] (AB239075)

Flow cytometry staining of human monocyte-derived dendritic cells (top) or human monocyte-derived dendritic cells treated with 1μg/mL lipopolysaccharide (LPS) for 24h (bottom), with ab239075 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). Monocyte-derived dendritic cells were incubated for 30 min at 4°C in 1x PBS containing 10 μg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab239075 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 106 in 100μl at 0.04 μg/ml (1/56500)) for 30min on ice. The cells were simultaneously stained with CD209.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 4°C

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on viable cells.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD86 antibody [EPR21962] (AB239075)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Daudi (human Burkitt's lymphoma cell line) cells labeling CD86 with ab239075 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in Daudi cell line is observed.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD86 antibody [EPR21962] (AB239075)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (human Burkitt's lymphoma cell line) cells labeling CD86 with ab239075 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in Raji cell line is observed.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD86 antibody [EPR21962] (AB239075)

Flow cytometric analysis of Raji (human Burkitt's lymphoma cell line) cell line labeling CD86 with ab239075 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

• IP

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Immunoprecipitation - Anti-CD86 antibody [EPR21962] (AB239075)

CD86 was immunoprecipitated from 0.35 mg of Raji whole (human Burkitt's lymphoma cell line) cell lysate with ab239075 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab239075 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : Raji whole cell lysate 10 μg (Input).

Lane 2 : ab239075 IP in Raji whole lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab239075 in Raji whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-CD86 antibody [EPR21962] (ab239075)

Predicted band size: 38 kDa

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• WB

Lab

Western blot - Anti-CD86 antibody [EPR21962] (AB239075)

Lanes 1 - 2 : Merged signal (red and green). Green - ab239075 observed at 70 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab239075 was shown to react with CD86 in wild-type Raji cells in western blot with loss of signal observed in CD86 knockout sample. Wild-type and CD86 knockout Raji cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab239075 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD86 antibody [EPR21962] (ab239075) at 1/1000 dilution

Lane 1:

Wild-type Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

CD86 knockout Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CD86 knockout Raji cell line (<a href='/ko/products/cell-lines/human-cd86-knockout-raji-cell-line-ab273858'>ab273858</a>)

Predicted band size: 38 kDa

Observed band size: 70 kDa

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• WB

Supplier Data

Western blot - Anti-CD86 antibody [EPR21962] (AB239075)

Exposure time : Lane 1 : 3 minutes; Lanes 2-3 : 26 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CD86 antibody [EPR21962] (ab239075) at 1/1000 dilution

Lane 1:

Ramos (human Burkitt's lymphoma cell line) whole cell lysate at 10 µg

Lane 2:

Raji (human Burkitt's lymphoma cell line) whole cell lysate at 10 µg

Lane 3:

Daudi (human Burkitt's lymphoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 38 kDa

Observed band size: 80 kDa

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