人IL1B knockout THP-1 cell line

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Human IL1B knockout THP-1 cell line (AB273762)

ab254360 staining IL-1 beta in wild-type THP-1 cells (top panel) and IL1B knockout THP-1 cells (ab273762) (bottom panel) +/- TPA (80nM overnight) and LPS (100ng/ml, 6h). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab254360 at 0.4μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• WB

Lab

Western blot - Human IL1B knockout THP-1 cell line (AB273762)

Western blot : Anti-IL1B antibody [RM1009] (ab283818) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab283818 was shown to bind specifically to IL1B. A band was observed at 34 kDa in wild-type THP-1 cell lysates with no signal observed at this size in IL1B knockout cell line. To generate this image, wild-type and IL1B knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-IL-1 beta antibody [RM1009] (<a href='/products/primary-antibodies/il-1-beta-antibody-rm1009-ab283818'>ab283818</a>) at 1/1000 dilution

Lane 1:

Wild-type THP-1 Vehicle control: TPA (80 nM overnight) then LPS (0 ng/mL, 6 h) cell lysate at 20 µg

Lane 2:

Wild-type THP-1 TPA (80 nM overnight) then LPS (100 ng/mL, 6 h) cell lysate at 20 µg

Lane 3:

IL-1b knockout THP-1 Vehicle control: TPA (80 nM overnight) then LPS (0 ng/mL, 6 h) cell lysate at 20 µg

Lanes 3 - 4:

Western blot - Human IL1B knockout THP-1 cell line (ab273762)

Lane 4:

IL-1b knockout THP-1 TPA (80 nM overnight) then LPS (100 ng/mL, 6 h) cell lysate at 20 µg

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• WB

Lab

Western blot - Human IL1B knockout THP-1 cell line (AB273762)

Lanes 1 - 4 : Merged signal (red and green). Green - ab216995 observed at 27-32 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab216995 was shown to react with IL-1 beta in LPS-treated wild-type THP-1 cells in Western blot with loss of signal observed in IL1B knockout cell line ab273762 (LPS-treated) (knockout cell lysate ab275508). Wild-type THP-1 and IL1B knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab216995 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 ° 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 h at room temperature before imaging.

All lanes:

Western blot - Anti-IL-1 beta antibody [EPR21086] (<a href='/products/primary-antibodies/il-1-beta-antibody-epr21086-ab216995'>ab216995</a>) at 1/1000 dilution

Lane 1:

Wild-type THP-1 untreated cell lysate at 30 µg

Lane 2:

Wild-type THP-1 LPS-treated (3 h, 100 ng/ml) cell lysate at 30 µg

Lane 2:

Western blot - Human IL1B knockout THP-1 cell line (ab273762)

Lane 3:

IL1B knockout THP-1 untreated cell lysate at 30 µg

Lane 4:

IL1B knockout THP-1 LPS-treated (3 h, 100 ng/ml) cell lysate at 30 µg

Predicted band size: 30 kDa

Observed band size: 27-32 kDa

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• sELISA

Lab

Sandwich ELISA - Human IL1B knockout THP-1 cell line (AB273762)

Human IL-1 beta concentration was interpolated from the standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human IL-1 beta ELISA kit (ab229384). Wild-type and IL-1 beta knockout THP-1 cells (ab273762) were assessed in duplicate (n=2). Cells were either treated with LPS (100 ng/ml, 3 h) then ATP (5 mM, 45 min) to induce expression of IL-1 beta or not treated. Data are represented as the mean and error bars represent standard deviation.

• Sanger seq

Supplier Data

Sanger Sequencing - Human IL1B knockout THP-1 cell line (AB273762)

Allele-1 : 47 bp deletion in exon 4.