Human IL1B knockout THP-1 cell line
- Advanced Validation
- What is this?
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(0 Publication)
- 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)
All lanes:
Western blot - Anti-IL-1 beta antibody [RM1009] (<a href='/en-us/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
false
- 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='/en-us/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
false
- 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.
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Reactivity data
Product details
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
Handling procedures
Initial handling guidelines
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 4x105 cells/mL. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.
5. THP-1 cells recover slowly from cryopreservation and therefore may not be ready for subculture for a number of days. Cells should be left as much as possible over this time and only subcultured when the cell density reaches 8x105 cells/mL.Small amounts of fresh media can be added until cell number/viability improves.
Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- Cells should be seeded at 2x105 - 3x105 cells/mL and subcultured when they have reached 8x105 cells/mL.
- It is not recommended to allow the cell density to exceed 1x106 cells/mL.
Culture medium
RPMI + 10% FBS + 0.05 mM beta-mercaptoethanol
Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Suspension
Gender
Male
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com