IL1RN KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 49 bp deletion in exon 5.
Images
Key facts
- Cell type
- A-431
- Species or organism
- Human
- Tissue
- Skin
- Form
- Liquid
- Knockout validation
- Immunocytochemistry, Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 49 bp deletion in exon 5.
Alternative names
ICIL-1RA, IL-1ra3, IL1F3, IRAP, MGC10430, intracellular IL-1 receptor antagonist type II, intracellular interleukin-1 receptor antagonist, type II interleukin-1 receptor antagonist
Recommended products
IL1RN KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 49 bp deletion in exon 5.
Key facts
- Cell type
- A-431
- Species or organism
- Human
- Tissue
- Skin
- Form
- Liquid
- Knockout validation
- Immunocytochemistry, Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 49 bp deletion in exon 5.
- Disease
- Epidermoid Carcinoma
- Concentration
Properties
- Gene name
- IL1RN
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Immunocytochemistry, Sanger Sequencing, Western blot
- Zygosity
- Homozygous
Quality control
- STR analysis
- CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
- EU: 1 US: 1
- Adherent/suspension
- Adherent
- Gender
- Female
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 seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.- Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
- Culture medium
- DMEM (High Glucose) + 10% FBS
- Cryopreservation medium
- Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Storage
- Shipped at conditions
- Dry Ice
- Appropriate short-term storage conditions
- -196°C
- Appropriate long-term storage conditions
- -196°C
Notes
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.
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6 product images
Immunocytochemistry - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
IL-1RA staining observed in wild-type A431 cells (top panel) and IL1RN knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 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 an anti-IL-1ra antibody at 10μg/ml concentration (shown in green) and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 (Mouse monoclonal to alpha Tubulin - Alexa Fluor® 594) at 1/250 dilution (shown in red) overnight at 4°C. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
False colour image of Western blot: Anti-IL-1RA antibody [EPR6483] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-IL-1RA antibody [EPR6483] ab124962 was shown to bind specifically to IL-1RA. A band was observed at 19 kDa in wild-type A431 cell lysates with no signal observed at this size in IL1RN knockout cell line ab273379 (knockout cell lysate Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell lysate ab275530). To generate this image, wild-type and IL1RN knockout A431 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 (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-IL-1RA antibody [EPR6483] (Anti-IL-1RA antibody [EPR6483] ab124962) at 1/10000 dilution
Lane 1: Wild-type A431 cell lysate at 20 µg
Lane 2: IL-1RA knockout A431 cell lysate at 20 µg
Lane 2: Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
Performed under reducing conditions.
Predicted band size: 20 kDa
Observed band size: 19 kDa
Sandwich ELISA - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
Human IL-1RA concentration was interpolated from the IL-1RA standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human IL-1ra ELISA Kit (Human IL-1ra ELISA Kit ab211650). Wild-type A-431, IL1RN knockout A-431 (ab273379) and THP-1 cells were assessed in duplicate (n=2). Data are represented as the mean and error bars represent standard deviation.
Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-IL-1ra antibody observed at 18 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
Anti-IL-1ra antibody was shown to react with IL-1ra in wild-type A431 cells in Western blot with loss of signal observed in IL-1RA knockout cell line ab273379 (IL-1RA knockout cell lysate Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell lysate ab275530). Wild-type A431 and IL-1RA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with anti-IL-1ra antibody and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at 0.5 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Donkey anti-Goat IgG H&L (IRDye® 800CW) preabsorbed (Donkey anti-Goat IgG H&L (IRDye® 800CW) preadsorbed ab216775) and Donkey anti-Mouse 680RD secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.All lanes: Anti-IL-1ra antibody at 0.5 µg/mL
Lane 1: Wild-type A431 cell lysate at 20 µg
Lane 2: IL-1RA knockout A431 cell lysate at 20 µg
Lane 2: Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
Lane 3: HeLa cell lysate at 20 µg
Lane 4: SK-OV-3 cell lysate at 20 µg
Performed under reducing conditions.
Sanger Sequencing - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
49 bp deletion in exon 5
Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
All lanes: Western blot - Anti-IL-1RA antibody [EPR6483] (Anti-IL-1RA antibody [EPR6483] ab124962) at 1/50000 dilution
Lane 1: Wild-type A431 cell lysate at 20 µg
Lane 2: IL1RN knockout A431 cell lysate at 20 µg
Lane 2: Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
Lane 3: MOLT-4 cell lysate at 20 µg
Lane 4: Human Kidney cell lysate at 20 µg
SecondaryLanes 1 - 4: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution
Lanes 1 - 4: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Observed band size: 18 kDa
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