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AB281618

Mouse SIRPA knockout RAW 264.7 cell line

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SIRPA KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, Homozygous: 35 bp deletion in exon 6. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
3 Images
Western blot - Mouse SIRPA knockout RAW 264.7 cell line (AB281618)
  • WB

Lab

Western blot - Mouse SIRPA knockout RAW 264.7 cell line (AB281618)

False colour image of Western blot : Anti-SIRP alpha antibody [EPR16264] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab191419 was shown to bind specifically to SIRP alpha. A band was observed at 100-140 kDa (mouse SIRPA, isoform 1) & 40-50 kDa (mouse SIRPA, isoform 2), in wild-type RAW 264.7 cell lysates (band observed at 70-100 kDa in THP-1 is Human SIRPA) with no signal observed at this size in SIRPA knockout cell line ab281618 (knockout cell lysate ab282969). To generate this image, wild-type and SIRPA knockout RAW 264.7 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

observed band : 100-140 kDa (mouse SIRPA, isoform 1) & 40-50 kDa (mouse SIRPA, isoform 2)

All lanes:

Western blot - Anti-SIRP alpha antibody [EPR16264] (<a href='/en-us/products/primary-antibodies/sirp-alpha-antibody-epr16264-ab191419'>ab191419</a>) at 1/1000 dilution

Lane 1:

Wild-type RAW 264.7 cell lysate at 20 µg

Lane 2:

SIRPA knockout RAW 264.7 cell lysate at 20 µg

Lane 2:

Western blot - Mouse SIRPA knockout RAW 264.7 cell line (ab281618)

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Predicted band size: 55 kDa

Observed band size: 100-140 kDa

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Western blot - Mouse SIRPA knockout RAW 264.7 cell line (AB281618)
  • WB

Lab

Western blot - Mouse SIRPA knockout RAW 264.7 cell line (AB281618)

False colour image of Western blot : Anti-SIRPA antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to SIRPA. A band was observed at 100-140 kDa (mouse SIRPA, isoform 1), in wild-type RAW 264.7 cell lysates (band observed at 70-100 kDa in THP-1 is Human SIRPA) with no signal observed at this size in SIRPA knockout cell line ab281618 (knockout cell lysate ab282969). To generate this image, wild-type and SIRPA knockout RAW 264.7 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (<a href='/en-us/products/primary-antibodies/gapdh-antibody-6c5-loading-control-ab8245'>ab8245</a>) at 1/20000 dilution

Lane 1:

Wild-type RAW 264.7 cell lysate at 20 µg

Lane 2:

RAW 264.7 cell lysate at 20 µg

Lane 2:

Western blot - Mouse SIRPA knockout RAW 264.7 cell line (ab281618)

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Predicted band size: 55 kDa

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Sanger Sequencing - Mouse SIRPA knockout RAW 264.7 cell line (AB281618)
  • Sanger seq

Supplier Data

Sanger Sequencing - Mouse SIRPA knockout RAW 264.7 cell line (AB281618)

35 bp deletion in exon 6

Key facts

Cell type

RAW 264.7

Species or organism

Mouse

Tissue

Lymphatic

Form

Liquid

form

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: 35 bp deletion in exon 6

Disease

Carcinoma

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Primary Antibodies

AB191419

Anti-SIRP alpha antibody [EPR16264]

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Primary Antibodies

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Anti-LILRB1 antibody [EPR21007]

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LILRB1 antibody [EPR21007] (AB229186)

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Reactivity data

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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.

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What's included?

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Properties and storage information

Gene name
SIRPA
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Zygosity
Homozygous
Shipped at conditions
Dry Ice
Appropriate short-term storage conditions
-196°C
Appropriate long-term storage conditions
-196°C
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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 for bath 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 all remaining cells into an ultra-low attachment 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.
5. Once confluent passage into an appropriate flask at a density of 2x105 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
  • Grow in ultra-low attachment flasks.
  • Do not wash or use dissociation reagent. Cells are semi-adherent. Tap flask sharply to remove adhered cells.
  • All seeding densities should be based on cell counts gained by established methods.
  • A guide seeding density of 2x105 cells/mL is recommended.
  • Cells should be passaged when they have achieved 80-90% confluence.
Culture medium

DMEM + 10% FBS

Cryopreservation medium

Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

SIRP alpha also known as CD172a is a transmembrane receptor protein with a mass ranging between 70-110 kDa due to glycosylation. This protein extensively expresses on the surface of myeloid cells neurons and a subset of T-cells and is part of the immunoglobulin superfamily. SIRP alpha interacts with its ligand CD47 a widely expressed glycoprotein involved in immune response regulation. Its mechanical action primarily involves signal regulation through the recruitment of SHP-1 and SHP-2 two cytoplasmic tyrosine phosphatases.
Biological function summary

SIRP alpha functions significantly in the regulation of phagocytosis acting as a "don't eat me" signal to macrophages upon binding with CD47. It does not act alone; rather it is part of a complex that recruits SHP-1 and SHP-2 leading to inhibition of dephosphorylation activities essential for engulfment processes. This regulatory mechanism is important for maintaining cellular homeostasis ensuring that healthy cells are not mistakenly destroyed by the immune system.

Pathways

SIRP alpha plays an important role in the innate immune pathways involving the regulation of phagocytosis and cell-cell adhesion. Particularly it fits into the immune checkpoint pathways where it interacts closely with proteins like CD47 and plays a role in the interaction between the immune system and cancer cells. Through these pathways SIRP alpha helps maintain balance in the immune response allowing for the recognition of self versus non-self therefore preventing autoimmunity while facilitating the clearance of pathogens.

SIRP alpha is implicated in cancer and autoimmune diseases. In cancer its interaction with CD47 allows tumor cells to escape phagocytosis promoting tumor survival and growth. In autoimmune disorders dysregulated SIRP alpha expression or signaling could miscommunicate immune signals leading to the destruction of healthy tissue. Understanding the link between SIRP alpha and these conditions can reveal potential targets for therapeutic development especially using inhibitors or modulators targeting the SIRP alpha-CD47 interaction.
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Cell culture

Biosafety level

EU: 2 US: 2

Adherent/suspension

Adherent

Gender

Male

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Product protocols

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Target data

See full target information SIRPA
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Product promise

We are committed to supporting your work with high-quality reagents, and we're here for you every step of the way. In the unlikely event that one of our products does not perform as expected, you're protected by our Product Promise.
For full details, please see our Terms & Conditions

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