Human SYK knockout THP-1 cell line (ab288700)

SYK 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: 107 bp deletion in exon 2.

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Images

Immunocytochemistry/ Immunofluorescence - Human SYK knockout THP-1 cell line (AB288700), expandable thumbnail

Key facts

Cell type: THP-1
Species or organism: Human
Tissue: Blood
Form: Liquid
Knockout validation: Immunocytochemistry, Sanger Sequencing, Western blot
Mutation description: Knockout achieved by using CRISPR/Cas9, Homozygous: 107 bp deletion in exon 2

Alternative names

EC 2.7.10.2, KSYK_HUMAN, Spleen tyrosine kinase, Tyrosine-protein kinase SYK, kinase Syk, p72-Syk

Key facts

Cell type: THP-1
Species or organism: Human
Tissue: Blood
Form: Liquid
Knockout validation: Immunocytochemistry, Sanger Sequencing, Western blot
Mutation description: Knockout achieved by using CRISPR/Cas9, Homozygous: 107 bp deletion in exon 2
Disease: Acute Monocytic Leukemia
Concentration

Properties

Gene name: SYK
Gene editing type: Knockout
Gene editing method: CRISPR technology
Knockout validation: Immunocytochemistry, Sanger Sequencing, Western blot
Zygosity: Homozygous

Quality control

STR analysis: VWA, TPOX, CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539

Cell culture

Biosafety level: EU: 1 US: 1
Adherent/suspension: Suspension
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. 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.

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 THP-1 cell line (ab281894). 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.

Upon thawing make banks as soon as possible and use each bank within 10-20 passages

As cell line growth can vary please attempt culture for 2 weeks from revival of initial bank before contacting the technical team.

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

Spleen tyrosine kinase commonly known as Syk is a non-receptor tyrosine kinase with a mass of about 72 kDa. It serves as a critical signaling molecule in immune cells. Syk is expressed in various cell types such as B cells T cells monocytes and others involved in the immune response. It initiates signaling cascades by binding to immunoreceptor tyrosine-based activation motifs (ITAMs). Alternate forms include phosphorylated Syk often referred to as p-Syk which activates various downstream signaling pathways.

Biological function summary

Syk plays a role in transmitting signals from the cell surface to the interior of the cell facilitating various cellular activities. It is a part of signal transduction complexes and functions in the regulation of immune cell activation differentiation and survival. It affects processes like Fc receptor signaling in macrophages and neutrophils impacting immune responses and inflammation. The phospho-Syk form influences the strength and duration of signaling affecting various cellular functions.

Pathways

Syk is significant in the B-cell receptor (BCR) signaling pathway and Fc receptor signaling pathway. Within these pathways Syk interacts with proteins like Lyn and Fyn which are other kinases involved in BCR signal transduction. Syk phosphorylation noted as p-Syk plays a part in activating downstream molecules such as phospholipase C gamma (PLCγ) to mediate cellular responses to external stimuli. The critical role of Syk in these pathways influences both adaptive and innate immune responses.

Associated diseases and disorders

Syk correlates with conditions like rheumatoid arthritis and certain B-cell lymphomas. In rheumatoid arthritis aberrant Syk signaling contributes to the inflammation and joint destruction characteristic of the disease. Syk's role in B-cell antigen receptor signaling links it to B-cell lymphomas where dysregulation in signaling pathways leads to uncontrolled proliferation of malignant B cells. The involvement of Syk in both disease contexts suggests its potential as a therapeutic target along with pathways related to proteins like BLNK and PLCγ in these disorders.

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10 product images

Immunocytochemistry/ Immunofluorescence - Human SYK knockout THP-1 cell line (ab288700)
Anti-Syk antibody [EPR573-69] - BSA and Azide free (Detector) ab244968 was shown to react with SYK in wild-type THP-1 cells in immunocytochemistry with loss of signal observed in a SYK knockout cell line. Anti-Syk antibody [EPR573-69] - BSA and Azide free (Detector) ab244968 at 1/1000 dilution.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
Western blot - Human SYK knockout THP-1 cell line (ab288700)
Anti-Syk antibody [EPR573-69] - BSA and Azide free (Detector) ab244968 was shown to react with SYK in wild-type THP-1 cells in Western blot with loss of signal observed in SYK knockout cell line ab288700. Wild-type THP-1 and SYK knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with Anti-Syk antibody [EPR573-69] - BSA and Azide free (Detector) ab244968 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
All lanes: Western blot - Anti-Syk antibody [EPR573-69] - BSA and Azide free (Detector) (Anti-Syk antibody [EPR573-69] - BSA and Azide free (Detector) ab244968) at 1/1000 dilution
Lane 1: Wild-type THP-1 lysate at 30 µg
Lane 2: SYK knock-out THP-1 lysate at 30 µg
Western blot - Human SYK knockout THP-1 cell line (ab288700)
Western blot: Anti-SYK antibody [EP573Y] (Anti-Syk antibody [EP573Y] ab40781) staining at 1/1000 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-Syk antibody [EP573Y] ab40781 was shown to bind specifically to SYK. A band was observed at 72 kDa in wild-type THP-1 cell lysates with no signal observed at this size in SYK knockout cell line ab288700 (knockout cell lysate ab289593). To generate this image, wild-type and SYK 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 5 % 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-Syk antibody [EP573Y] (Anti-Syk antibody [EP573Y] ab40781) at 1/1000 dilution
Lanes 1 - 4: Western blot at 20 µg
Lane 2: Western blot - Human SYK knockout THP-1 cell line (ab288700)
Secondary
All 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: 72 kDa
Observed band size: 72 kDa
Sanger Sequencing - Human SYK knockout THP-1 cell line (ab288700)
127 bp deletion in exon 2.
Western blot - Human SYK knockout THP-1 cell line (ab288700)
All lanes: Western blot - Anti-Syk antibody [SYK-01] (Anti-Syk antibody [SYK-01] ab3993) at 1/500 dilution
Lane 1: Wild-type THP-1 lysate at 20 µg
Lane 2: Western blot - Human SYK knockout THP-1 cell line (ab288700) at 20 µg
Observed band size: 72 kDa
Western blot - Human SYK knockout THP-1 cell line (ab288700)
All lanes: Western blot - Anti-Syk antibody [EPR19414-176] - BSA and Azide free (Capture) (Anti-Syk antibody [EPR19414-176] - BSA and Azide free (Capture) ab244701) at 1/1000 dilution
Lane 1: Wild-type THP-1 lysate at 20 µg
Lane 2: Western blot - Human SYK knockout THP-1 cell line (ab288700) at 20 µg
Observed band size: 72 kDa
Immunocytochemistry/ Immunofluorescence - Human SYK knockout THP-1 cell line (ab288700)
Immunocytochemistry/ Immunofluorescence - Human SYK knockout THP-1 cell line (ab288700)
Western blot - Human SYK knockout THP-1 cell line (ab288700)
All lanes: Western blot - Anti-Syk antibody [EP573Y] (Anti-Syk antibody [EP573Y] ab40781) at 1/1000 dilution
Lane 1: Wild-type THP-1 lysate at 20 µg
Lane 2: Western blot - Human SYK knockout THP-1 cell line (ab288700) at 20 µg
Observed band size: 72 kDa
Immunocytochemistry/ Immunofluorescence - Human SYK knockout THP-1 cell line (ab288700)