Human CXCL8 knockout PC-3 cell line (ab273743)
Overview
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Product name
Human CXCL8 knockout PC-3 cell line -
Parental Cell Line
PC3 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 22% 11 bp deletion, 24% 7 bp deletion, 54% 2 bp deletion in exon 2 -
Passage number
<20 -
Knockout validation
Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: WB, Sandwich ELISAmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type PC3 cell line (ab275472). 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.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: F-12K + 10% FBS
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 2x104 cells/cm2. 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.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.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Prostate -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Male -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell types in response to an inflammatory stimulus. IL-8(6-77) has a 5-10-fold higher activity on neutrophil activation, IL-8(5-77) has increased activity on neutrophil activation and IL-8(7-77) has a higher affinity to receptors CXCR1 and CXCR2 as compared to IL-8(1-77), respectively. -
Sequence similarities
Belongs to the intercrine alpha (chemokine CxC) family. -
Post-translational
modificationsSeveral N-terminal processed forms are produced by proteolytic cleavage after secretion from at least peripheral blood monocytes, leukcocytes and endothelial cells. In general, IL-8(1-77) is referred to as interleukin-8. IL-8(6-77) is the most promiment form. -
Cellular localization
Secreted. - Information by UniProt
Associated products
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Biochemicals
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KO cell lysates
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Related Products
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SimpleStep ELISA kits
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab273743 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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WB |
Use at an assay dependent concentration.
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Sandwich ELISA |
Use at an assay dependent concentration.
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Notes |
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WB
Use at an assay dependent concentration. |
Sandwich ELISA
Use at an assay dependent concentration. |
Images
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All lanes : Anti-IL-8 antibody [EPR22994-255] (ab235584) at 1/1000 dilution
Lane 1 : Wild-type PC-3 Brefeldin A (ab120299)-treated (5 μg/ml, 5 h) cell lysate
Lane 2 : Wild-type PC-3 LPS-treated (2 μg/ml, 6 h) with Brefeldin A (ab120299) (5 μg/ml, 5 h) cell lysate
Lane 3 : CXCL8 knockout PC-3 Brefeldin A (ab120299)-treated (5 μg/ml, 5 h) cell lysate
Lane 4 : CXCL8 knockout PC-3 LPS-treated (2 μg/ml, 6 h) with Brefeldin A (ab120299) (5 μg/ml, 5 h) cell lysate
Lane 5 : A431 cell lysate
Lane 6 : HCT116 cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Observed band size: 10 kDa why is the actual band size different from the predicted?Lanes 1 - 6: Merged signal (red and green). Green - ab235584 observed at 10 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab235584 was shown to react with IL-8 in wild-type PC-3 cells in Western blot with loss of signal observed in CXCL8 knockout cell line ab273743 (knockout cell lysate ab275520). Wild-type PC-3 and CXCL8 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab235584 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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 (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.
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Human IL-8 concentration was interpolated from the standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human IL-8 ELISA kit (ab214030). Wild-type PC-3 cells and CXCL8 knockout PC-3 cells (ab273743) were assessed in duplicate (n=2) and were either treated with 2 µg/ml LPS for 6 hours to induce expression of IL-8 or not treated with LPS. Data are represented as the mean and error bars represent standard deviation.
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Allele-1: 11bp deletion in exon 2.
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Allele-2: 7bp deletion in exon 2.
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Allele-3: 2bp deletion in exon 2.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab273743 has not yet been referenced specifically in any publications.