HEK-Dual HTLR5 (NF/IL8) | Data Sheet | InvivoGen

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HEK-Dual™ hTLR5 (NF/IL8) Cells
(NF-kB-SEAP/KI-[IL-8]Lucia)
Dual NF-kB and IL-8 reporter HEK293 cells expressing human TLR5
Catalog # hkd-htlr5ni
http://www.invivogen.com/hek-dual-htlr5
For research use only
Version # 17C29-MM
PRODUCT INFORMATION
Content
• 1 vial of HEK-Dual™ hTLR5 (NF/IL8) Cells (3-7 x 106 cells)
• 100 µl Hygromycin B Gold (100 mg/ml). Store at 4 °C or at -20°C.*
• 100 µl Zeocin™ (100 mg/ml). Store Zeocin™ at 4 °C or at -20°C.*
• 1 ml Normocin™ (50 mg/ml). Normocin™ is a formulation of 3 antibiotics
active against mycoplasmas, bacteria and fungi. Store at -20°C.*
*The expiry date is specified on the product label.
• 1 pouch of QUANTI-Blue™ (SEAP detection medium)
Store QUANTI-Blue™ pouch at 4 °C for 6 months. Reconstituted
QUANTI-Blue™ medium is stable for 2 weeks at 4 °C. Protect from light.
• 1 pouch of QUANTI-Luc™ (Lucia luciferase detection medium)
Store QUANTI-Luc™ pouch at -20°C for 12 months. Reconstituted
QUANTI-Luc™ medium is stable for 1 week at 4 °C and for 1 month
at -20°C. Protect QUANTI-Luc™ from light.
Handling Cells Upon Receipt
Cells must be thawed immediately upon receipt and grown according to
handling procedures (see next page), to ensure cell viability and proper
assay performance.
Note: Do not freeze the cells upon receipt as it may result in irreversible
damage to the cell line.
Disclaimer: We cannot guarantee cell viability if the cells are not thawed
immediately upon receipt and grown according to handling procedures.
Cell Line Stability
Genetic instability is a biological phenomenon that occurs in all stably
transfected cells. Cells will undergo genotypic changes resulting in
reduced responsiveness over time in normal cell culture conditions.
Therefore, it is critical to prepare an adequate number of frozen stocks at
early passages. To ensure maximum efficiency, do not passage
HEK-Dual™ hTLR5 (NF/IL8) cells more than 15 times. These cells should
be maintained in growth medium supplemented with selective antibiotics,
Hygromycin B Gold (100 mg/ml) and Zeocin™ (50 mg/ml).
Quality Control
• The biallelic replacement of the human interleukin-8 (IL-8) open
reading frame (ORF) with the Lucia luciferase reporter ORF was verified
by PCR and sequencing. Furthermore, the inability to produce IL-8 has
been confirmed by ELISA.
• TLR3 and TNF receptor (TNFR) knockout has been verified by
functional assays and DNA sequencing to confirm frameshift deletion.
• The expression of the human TLR5 (hTLR5) gene has been confirmed
by RT-PCR.
• The response to various TLR agonists has been tested. As expected, only
TLR5 agonists induced the production of the reporter proteins.
• The cell line stability for 15 passages following thawing has been verified.
• The cell line is guaranteed mycoplasma-free.
BACKGROUND
HEK-Dual™ TLR (NF/IL8) cells are a family of Toll-like receptor (TLR)reporter cells designed for the study of the inflammatory response induced
by the stimulation of a given TLR by monitoring the activation of the
transcription factors NF-kB and AP-1 and/or the expression of
interleukin-8 (IL-8). IL-8 is a chemokine produced in response to TLR
agonists in an NF-kB and AP-1 dependent-manner1, 2. HEK-Dual™ TLR
(NF/IL8) cells were generated from the human embryonic kidney 293
(HEK293)-derived cell line, HEK-Dual™ Null (NF/IL8), by stable
transfection of a TLR gene. This parental cell line, that features a triple
knockout of TLR3, TLR5 and TNFR (all of which are endogenously
expressed in HEK293 cells), stably expresses an NF-kB/AP-1 inducible
secreted embryonic alkaline phosphatase (SEAP) reporter construct. It
also expresses Lucia luciferase, a secreted luciferase, placed under the
control of the endogenous IL-8 promoter; the coding sequence of IL-8
was replaced by the Lucia luciferase ORF using knockin technology.
Thus, TLR stimulation can be assessed in HEK-Dual™ TLR (NF/IL8)
cells by monitoring NF-kB/AP-1-induced SEAP production and/or IL-8dependent expression of Lucia luciferase.
The two reporter proteins, SEAP and Lucia Luciferase, can be readily
measured in the supernatant by using QUANTI-Blue™ and
QUANTI-Luc™, respectively.
1. Ohta K. et al., 2014. Toll-like receptor (TLR) expression and
TLR-mediated interleukin-8 production by human submandibular gland
epithelial cells. Mol Med Rep. 10(5):2377-82. 2. Roebuck KA. et al., 1999.
Regulation of interleukin-8 gene expression. J Interferon Cytokine Res.
19(5):429-38.
CELL LINE DESCRIPTION
HEK-Dual™ TLR5 (NF/IL8) cells were generated from HEK-Dual™ Null
cells by stable transfection of the human TLR5 (hTLR5) gene. Due to the
knockout of TLR3, these cells enable the study of hTLR5 signaling without
interference from other TLRs. They respond to low concentrations of
TLR5 agonist flagellin. They do not respond to other TLR agonists or to
the cytokine TNF-α (see validation sheet).
Note: HEK-Dual™ hTLR5 (NF/IL8) and their parental cell line
endogenously express NOD1.
HEK-Dual™ hTLR5 (NF/IL8) cells are resistant to hygromycin B,
Zeocin™ and blasticidin. They should be maintained in growth medium
(see next page) supplemented with hygromycin B and Zeocin™.
TECHNICAL SUPPORT
InvivoGen USA (Toll‑Free): 888-457-5873
InvivoGen USA (International): +1 (858) 457-5873
InvivoGen Europe: +33 (0) 5-62-71-69-39
InvivoGen Hong Kong: +852 3-622-34-80
E-mail: info@invivogen.com
www.invivogen.com
SAFETY CONSIDERATIONS
Biosafety Level 2
HEK-Dual™ hTLR5 (NF/IL8) cells were derived from HEK293 cells
(transformed with adenovirus 5 DNA) and thus may require Biosafety
Level 2. The biosafety level varies by country. In the United States,
HEK293 cell lines are designated Biosafety Level 2 according to the
Center for Disease Control and Prevention (CDC). In Germany, HEK293
cell lines are designated Biosafety Level 1 according to the Central
Committee of Biological Safety, Zentrale Kommission für die
Biologische Sicherheit (ZKBS). Please check with your country’s
regulatory authority regarding the use of these cells.
HANDLING PROCEDURES
Required Cell Culture Medium
• Growth Medium: DMEM, 4.5 g/l glucose, 10% (v/v) fetal bovine
serum (FBS), 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml
Normocin™, 2 mM L-glutamine
• Freezing Medium: DMEM with 20% (v/v) FBS and 10% (v/v) DMSO
• Test Medium: DMEM, 4.5 g/l glucose, 10% (v/v) heat-inactivated FBS
(30 min at 56°C), 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml
Normocin™, 2 mM L-glutamine
Note: Heat-inactivated FBS is also commercially available.
Required Selective Antibiotics
Hygromycin B Gold and Zeocin™
Initial Culture Procedure
The first propagation of cells should be for generating stocks for future
use. This ensures the stability and performance of the cells for subsequent
experiments.
1. Thaw the vial by gentle agitation in a 37 °C water bath. To reduce the
possibility of contamination, keep the O-ring and cap out of the water.
Thawing should be rapid.
2. Remove the vial from the water bath as soon as the contents are thawed,
and decontaminate by dipping in or spraying with 70% (v/v) ethanol.
Note: All steps from this point should be carried out under strict aseptic
conditions.
3. Transfer cells into a larger vial containing 15 ml of pre-warmed growth
medium. Do not add selective antibiotics until the cells have been
passaged twice.
4. Centrifuge vial at 1000-1200 RPM (RCF 200-300 g) for 5 minutes.
5. Remove supernatant containing the cryoprotective agent and
resuspend cells with 1 ml of growth medium without selective antibiotics.
6. Transfer the vial contents to a 25 cm2 tissue culture flask
containing 5 ml of growth medium without selective antibiotics.
7. Place the flask containing cells at 37 °C in 5% CO2.
Frozen Stock Preparation
1. Resuspend cells at a density of 5-7 x 106 cells/ml in freezing medium
freshly prepared with cold growth medium.
Note: A T-75 culture flask typically yields enough cells for preparing 3-4
frozen vials.
2. Dispense 1 ml of the cell suspension into cryogenic vials.
3. Place vials in a freezing container (Nalgene) and store at -80 °C overnight.
4. Transfer vials to liquid nitrogen for long term storage.
Note: If properly stored, cells should remain stable for years.
Cell Maintenance
1. After cells have recovered (after at least one passage), subculture the
cells in growth medium supplemented with 100 µg/ml of Hygromycin B
Gold and 50 µg/ml of Zeocin™.
2. Renew growth medium twice a week. Cells should be passaged when a
70-80% confluency is reached. Do not let the cell grow to 100% confluency.
Induction of HEK-Dual™ hTLR5 (NF/IL8) Cells
Day 1:
1. Add 20 ml of each sample per well of a flat-bottom 96-well plate.
2. Add 20 ml of a positive control such as FLA-ST (flagellin from
Salmonella typhimurium) at 100 ng/ml in one well.
3. Add 20 ml of a negative control such as sterile, endotoxin-free water in
another well.
4. Prepare a cell suspension of HEK-Dual™ hTLR5 (NF/IL8) cells
at ~280,000 cells per ml in test medium (containing 10% v/v
heat-inactivated FBS).
Note: Some FBS may contain alkaline phosphatases that can interfere
with SEAP quantification. We recommend to use heat-inactivated FBS to
inactivate these enzymes which are thermosensitive.
5. Add 180 ml of cell suspension (~50,000 cells) per well.
6. Incubate the plate at 37°C in a CO2 incubator for 20-24 h.
Detection of the NF-kB response using QUANTI-Blue™
Day 2:
1. Prepare QUANTI-Blue™ following the instructions on the enclosed
technical data sheet (TDS).
2. Add 180 ml of resuspended QUANTI-Blue™ per well of a
flat-bottom 96-well plate.
3. Add 20 ml of induced HEK-Dual™ hTLR5 (NF/IL8) cell culture supernatant.
4. Incubate the plate at 37°C incubator for 1-3 h.
5. Determine SEAP levels using a spectrophotometer at 620-655 nm.
Detection of the IL-8 response using QUANTI-Luc™
Below is a protocol for end-point readings using a luminometer, this
protocol can be adapted for use with kinetic measurements.
Day 2:
1. Prepare QUANTI-Luc™ following the instructions on the enclosed TDS.
2. Pipet 10 µl of HEK-Dual™ hTLR5 (NF/IL8) cell culture supernatant
per well in a 96-well white (opaque) or black plate, or a luminometer tube.
3. Add 50 μl of QUANTI-Luc™ per well.
4. Proceed immediately with the measurement.
USE RESTRICTIONS
These cells are distributed for research purposes only.
This product is covered by a Limited Use License. By use of this product,
the buyer agrees the terms and conditions of all applicable Limited Use
Label Licenses. For non-research use, such as screening, quality control
or clinical development, contact info@invivogen.com
RELATED PRODUCTS
Product Catalog Code
FLA-BS (Flagellin from B.subtilis) tlrl-bsfla
FLA-ST (Flagellin from S. typhimurium) tlrl-stfla
HEK-Dual™ Null Cells hkd-null
Hygromycin B Gold ant-hg-1
QUANTI-Blue™ rep-qb1
QUANTI-Luc™ rep-qlc1
RecFLA-ST (recombinant flagellin) tlrl-flic
Zeocin™ ant-zn-1
TECHNICAL SUPPORT
InvivoGen USA (Toll‑Free): 888-457-5873
InvivoGen USA (International): +1 (858) 457-5873
InvivoGen Europe: +33 (0) 5-62-71-69-39
InvivoGen Hong Kong: +852 3-622-34-80
E-mail: info@invivogen.com
www.invivogen.com

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