Human TNF knockout THP-1 cell line
Human TNF knockout THP-1 cell line
- Advanced Validation
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TNF 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: 52 bp deletion in exon 4. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
別名を表示する
TNF-a, TNFSF2, Tumor necrosis factor ligand superfamily member 2, Tumor necrosis factor, Cachectin, TNF-alpha, TNF, TNFA
- WB
Lab
Western blot - Human TNF knockout THP-1 cell line (AB273761)
Lanes 1 - 6 : Merged signal (red and green). Green - ab255275 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
ab255275 was shown to react with TNF alpha in THP-1 wild-type cells in Western blot with loss of signal observed in TNF knockout sample. Wild-type and TNF knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab255275 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 ° 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.
All lanes:
Western blot - Anti-TNF alpha antibody [EPR22598-212] (<a href='/products/primary-antibodies/tnf-alpha-antibody-epr22598-212-ab255275'>ab255275</a>) at 1/1000 dilution
Lane 1:
Wild-type THP-1 Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 2:
Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 2:
Western blot - Human TNF knockout THP-1 cell line (ab273761)
Lane 3:
TNF alpha knockout THP-1 Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 4:
TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 5:
U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 6:
U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Predicted band size: 25 kDa
Observed band size: 26 kDa
false
- WB
Lab
Western blot - Human TNF knockout THP-1 cell line (AB273761)
This Western blot image is a comparison between ab215188 and ab183218 tested under the same conditions. While ab215188 is suitable for WB for some samples ab183218 was found to be more sensitive. False colour image of Western blot : Anti-TNF alpha antibody [EPR20972] staining at 1/1000 dilution shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution shown in red. In Western blot ab215188 was shown to bind specifically to TNF alpha. A band was observed at 27 kDa in treated U937 cell lysates with no signal observed at this size without treatment. No signal was observed in wild-type THP-1 cell lysates or in TNF knockout cell line ab273761 (knockout cell lysate ab275507) with ab215188. However a band was observed at 27 kDa in treated wild-type THP-1 cell lysates with ab183218. To generate this image samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °. 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-TNF alpha antibody [EPR20972] (<a href='/products/primary-antibodies/tnf-alpha-antibody-epr20972-ab215188'>ab215188</a>) at 1/1000 dilution
Lane 1:
Wild-type THP-1 control: Brefeldin A (5 ug/mL, 4 h) cell lysate at 30 µg
Lane 2:
Wild-type treated THP-1: LPS (100 ng/mL, 16 h), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg
Lane 2:
Western blot - Human TNF knockout THP-1 cell line (ab273761)
Lane 3:
TNF alpha knockout THP-1 control: Brefeldin A (5 ug/mL, 4 h) cell lysate at 30 µg
Lane 4:
TNF alpha knockout THP-1 treated: LPS (100 ng/mL, 16 h), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg
Lane 5:
U937 control: PMA (10 mM, 2 days), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg
Lane 6:
U937 treated: PMA (10 mM, 2 days), LPS (1 ug/mL, last 16 h), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg
Predicted band size: 25 kDa
Observed band size: 27 kDa
false
- WB
Lab
Western blot - Human TNF knockout THP-1 cell line (AB273761)
Lanes 1 - 6 : Merged signal (red and green). Green - ab183218 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
ab183218 was shown to react with TNF alpha in THP-1 wild-type cells in Western blot with loss of signal observed in TNF knockout sample. Wild-type and TNF knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab183218 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 ° 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.
All lanes:
Western blot - Anti-TNF alpha antibody [EPR19147] (<a href='/products/primary-antibodies/tnf-alpha-antibody-epr19147-ab183218'>ab183218</a>) at 1/1000 dilution
Lane 1:
Wild-type THP-1 Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 2:
Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 2:
Western blot - Human TNF knockout THP-1 cell line (ab273761)
Lane 3:
TNF alpha knockout THP-1 Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 4:
TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 5:
U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Lane 6:
U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg
Predicted band size: 25 kDa
Observed band size: 26 kDa
false
- sELISA
Lab
Sandwich ELISA - Human TNF knockout THP-1 cell line (AB273761)
Human TNF alpha concentration was interpolated from the standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human TNF alpha ELISA kit (ab181421). Wild-type THP-1 and TNF alpha knockout THP-1 (ab273761) cells were assessed in duplicate (n=2). Cells were either treated with 100 ng/ml LPS for 16 h to induce expression of TNF alpha or not treated with LPS. Data are represented as the mean and error bars represent standard deviation."
- Sanger seq
Supplier Data
Sanger Sequencing - Human TNF knockout THP-1 cell line (AB273761)
Homozygous : 52 bp deletion in exon 4
組合せ製品
Reactivity data
製品の詳細
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.
製品内容
出荷温度及び保存条件
遺伝子名
遺伝子編集のタイプ
遺伝子編集の方法
ノックアウト検証方法
接合型
出荷温度
短期保存温度
長期保存温度
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Suspension
Gender
Male
初回取り扱いガイドライン
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. <p>1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.<br>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.<br>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 4x10<sup>5</sup> cells/mL. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.<br>4. Incubate the culture at 37°C incubator with 5% CO<sub>2</sub>. Cultures should be monitored daily.<br>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 8x10<sup>5</sup> cells/mL.Small amounts of fresh media can be added until cell number/viability improves.</p>
継代培養ガイドライン
- 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.
培養培地
RPMI + 10% FBS + 0.05 mM beta-mercaptoethanol
凍結保存培地
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
ターゲットの情報
Abcam product promise
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