IDO1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp deletion in exon 3.
Images
Key facts
- Cell type
A549
- Species or organism
Human
- Tissue
Lung
- Form
Liquid
- Knockout validation
Sanger Sequencing, Western blot
- Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp deletion in exon 3
Alternative names
3-dioxygenase, I23O1_HUMAN, IDO, IDO-1, INDO, Indole 2 3 dioxygenase, Indoleamine 2,3-dioxygenase 1, Indoleamine pyrrole 2 3 dioxygenase, Indoleamine-pyrrole 2, indolamine 2,3 dioxygenase, indoleamine 2 3 dioxygenase
Recommended products
IDO1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp deletion in exon 3.
Key facts
- Cell type
A549
- Species or organism
Human
- Tissue
Lung
- Form
Liquid
- Knockout validation
Sanger Sequencing, Western blot
- Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp deletion in exon 3
- Antibiotic resistance
Puromycin 1µg/mL
- Disease
Carcinoma
- Concentration
Properties
- Gene name
IDO1
- Gene editing type
Knockout
- Gene editing method
CRISPR technology
- Knockout validation
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
Male
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.
- Do not allow the cell density to exceed 7x104 cells/cm2.
- Culture medium
F-12K + 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
Recommended control: Human wild-type A549 cell line (Human wild-type A549 cell line ab255450). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
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.
Supplementary info
This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
Indoleamine 2 3-dioxygenase (IDO) also known as IDO1 is an enzyme involved in the catabolism of tryptophan to kynurenine. It weighs approximately 45 kDa and it is an important target in immunoregulatory processes. IDO is highly expressed in antigen-presenting cells such as dendritic cells and macrophages as well as in various tumor cells where it helps modulate immune responses. This enzyme can also be measured by using assays like IDO ELISA.
- Biological function summary
IDO plays a role in immune response regulation by degrading tryptophan an essential amino acid needed for T-cell proliferation. The depletion of tryptophan and the accumulation of its metabolites such as kynurenine cause immunosuppressive effects within the tissue microenvironment. While IDO functions mostly as a standalone enzyme its activity influences the cellular surroundings by altering the balance of local immune reactions.
- Pathways
IDO integrates into the tryptophan metabolism pathway and is important in the kynurenine pathway. Through this pathway it maintains immune homeostasis and cell defense mechanisms. Proteins like kynureninase and kynurenine 3-monooxygenase also participate in the same metabolic processes which further extend the effects of tryptophan metabolism on immune cell behavior.
- Associated diseases and disorders
IDO is linked with cancer and chronic inflammatory conditions. In cancer elevated IDO expression suppresses anti-tumor immunity aiding tumor cells to evade immune surveillance. It also plays a role in autoimmune disorders by regulating excessive immune activity. During cancer progression IDO often works in conjunction with immune checkpoint proteins like PD-L1 which further enhances its immunosuppressive capabilities within the tumor microenvironment.
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8 product images
Western blot - Human IDO1 (Indoleamine 2, 3-dioxygenase) knockout A549 cell line (ab266949)
Lanes 1 - 6:Merged signal (red and green). Green - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] ab76157 observed at 40 kDa. Red - loading control Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] ab76157 was shown to react with Indoleamine 2, 3-dioxygenase in treated wild-type A549 cells in Western blot with loss of signal observed in treated IDO1 knockout cell line ab266949 (IDO1 knockout cell lysate ab256948). Wild-type A549 and IDO1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] ab76157 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 2500 dilution and a 1 in 20000 dilution respectively. Blots were incubated with 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) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] ab76157) at 1/2500 dilution
Lane 1: Wild-type A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate at 20 µg
Lane 2: Wild-type A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg
Lane 3: IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate at 20 µg
Lane 4: IDO1 knockout A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg
Lane 5: SK-OV-3 cell lysate at 20 µg
Lane 6: MCF7 cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 45 kDa
Observed band size: 40 kDa
Western blot - Human IDO1 (Indoleamine 2, 3-dioxygenase) knockout A549 cell line (ab266949)
Lanes 1 - 6:Merged signal (red and green). Green - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] ab211017 observed at 40 kDa. Red - loading control Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] ab211017 was shown to react with Indoleamine 2, 3-dioxygenase in treated wild-type A549 cells in Western blot with no signal observed in treated IDO1 knockout cell line ab266949 (IDO1 knockout cell lysate ab256948). Wild-type A549 and IDO1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] ab211017 and Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 (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) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] ab211017) at 1/1000 dilution
Lane 1: Wild-type A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate at 20 µg
Lane 2: Wild-type A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg
Lane 3: IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate at 20 µg
Lane 4: IDO1 knockout A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg
Lane 5: SK-OV-3 cell lysate at 20 µg
Lane 6: MCF7 cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 45 kDa
Observed band size: 40 kDa
Western blot - Human IDO1 (Indoleamine 2, 3-dioxygenase) knockout A549 cell line (ab266949)
Lanes 1 - 6:Merged signal (red and green). Green - Anti-Indoleamine 2, 3-dioxygenase antibody [OTI1A3] ab156787 observed at 40 kDa. Red - loading control Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55 kDa.
Anti-Indoleamine 2, 3-dioxygenase antibody [OTI1A3] ab156787 was shown to react with Indoleamine 2, 3-dioxygenase in treated wild-type A549 cells in Western blot with no signal observed in treated IDO1 knockout cell line ab266949 (IDO1 knockout cell lysate ab256948). Wild-type A549 and IDO1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with Anti-Indoleamine 2, 3-dioxygenase antibody [OTI1A3] ab156787 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4 °C at a 1 in 4000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye®800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye®680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [OTI1A3] (Anti-Indoleamine 2, 3-dioxygenase antibody [OTI1A3] ab156787) at 1/4000 dilution
Lane 1: Wild-type A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate at 20 µg
Lane 2: Wild-type A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg
Lane 3: IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate at 20 µg
Lane 4: IDO1 knockout A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg
Lane 5: SK-OV-3 cell lysate at 20 µg
Lane 6: MCF7 cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 45 kDa
Observed band size: 40 kDa
Western blot - Human IDO1 (Indoleamine 2, 3-dioxygenase) knockout A549 cell line (ab266949)
Western blot: Anti-IDO1 antibody [SP260] (Anti-Indoleamine 2, 3-dioxygenase antibody [SP260] - C-terminal ab228468) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-Indoleamine 2, 3-dioxygenase antibody [SP260] - C-terminal ab228468 was shown to bind specifically to IDO1. A band was observed at 42 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IDO1 knockout cell line. To generate this image, wild-type and IDO1 knockout A549 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-Indoleamine 2, 3-dioxygenase antibody [SP260] - C-terminal (Anti-Indoleamine 2, 3-dioxygenase antibody [SP260] - C-terminal ab228468) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN gamma (25 ng/mL, 48 h) ab281500 cell lysate at 20 µg
Lane 2: Wild-type A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab255554 cell lysate at 20 µg
Lane 3: IDO1 knockout A549 Treated IFN gamma (25 ng/mL, 48 h) ab281489 cell lysate at 20 µg
Lane 4: IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab263515 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 45 kDa
Observed band size: 42 kDa
Western blot - Human IDO1 (Indoleamine 2, 3-dioxygenase) knockout A549 cell line (ab266949)
Western blot: Anti-IDO1 antibody [EPR24032-22] (Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] ab277522) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] ab277522 was shown to bind specifically to IDO1. A band was observed at 35/42 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IDO1 knockout cell line. To generate this image, wild-type and IDO1 knockout A549 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-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] (Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] ab277522) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN gamma (25 ng/mL, 48 h) ab281500 cell lysate at 20 µg
Lane 2: Wild-type A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab255554 cell lysate at 20 µg
Lane 3: IDO1 knockout A549 Treated IFN gamma (25 ng/mL, 48 h) ab281489 cell lysate at 20 µg
Lane 4: IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab263515 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 45 kDa
Observed band size: 35 kDa, 42 kDa
Sanger Sequencing - Human IDO1 (Indoleamine 2, 3-dioxygenase) knockout A549 cell line (ab266949)
Homozygous: 1 bp deletion in exon3
Western blot - Human IDO1 (Indoleamine 2, 3-dioxygenase) knockout A549 cell line (ab266949)
Western blot: Anti-IDO1 antibody [EPR1230Y] (Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] ab76157) staining at 1/2500 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] ab76157 was shown to bind specifically to IDO1. A band was observed at 42/56 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IDO1 knockout cell line. To generate this image, wild-type and IDO1 knockout A549 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-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] ab76157) at 1/2500 dilution
Lane 1: Wild-type A549 Treated IFN gamma (25 ng/mL, 48 h) ab281500 cell lysate at 20 µg
Lane 2: Wild-type A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab255554 cell lysate at 20 µg
Lane 3: IDO1 knockout A549 Treated IFN gamma (25 ng/mL, 48 h) ab281489 cell lysate at 20 µg
Lane 4: IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab263515 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 45 kDa
Observed band size: 42 kDa, 56 kDa
Western blot - Human IDO1 (Indoleamine 2, 3-dioxygenase) knockout A549 cell line (ab266949)
Western blot: Anti-IDO1 antibody [EPR28349-89] (Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] ab311847) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] ab311847 was shown to bind specifically to IDO1. A band was observed at 42 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IDO1 knockout cell line. To generate this image, wild-type and IDO1 knockout A549 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-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] ab311847) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN gamma (25 ng/mL, 48 h) ab281500 cell lysate at 20 µg
Lane 2: Wild-type A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab255554 cell lysate at 20 µg
Lane 3: IDO1 knockout A549 Treated IFN gamma (25 ng/mL, 48 h) ab281489 cell lysate at 20 µg
Lane 4: IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab263515 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 45 kDa
Observed band size: 42 kDa
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