HEK293 (ATCC CRL-1573) and BHK-21 (ATCC CCL-10) cells were cultured in Minimal Essential Media (MEM; Cellgro Mediatech, Inc, Manassas, VA USA), supplemented with 10% Fetal Bovine Serum (FBS; Corning, Corning, NY USA), 1× Penicillin/Steptomycin (Pen/Strep; Corning, Corning, NY USA), 1× Non-Essential Amino Acids (NEAA; Corning, Corning, NY USA), and l -glutamine (Corning, Corning, NY USA). HEK293-derived reporter cells, namely HEK-Blue hTLR3, HEK-Blue hTLR4, and HEK-Blue hTLR7 (Invivogen, San Diego, CA USA), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, Corning, NY USA) supplemented with 4.5 g/L glucose, 10% FBS, 1× Pen/Strep, and 1× Normocin (Invivogen, San Diego, CA USA). To maintain genetic homogeneity, the HEK-Blue tissue culture cells were maintained at low passage number and supplemented with the appropriate selection antibiotics on alternating passages to maintain genomic integrity (as indicated by Invivogen’s instructions per each cell line). All cell lines were cultured in humidified tissue culture incubators at 37 °C in the presence of 5.0% CO2.
Hek blue htlr7
Manufactured by InvivoGen
Sourced in United States
The HEK-Blue hTLR7 is a reporting cell line that stably expresses the human Toll-like receptor 7 (hTLR7) and a secreted embryonic alkaline phosphatase (SEAP) reporter gene. This cell line can be used to monitor the activation of hTLR7 in response to specific ligands.
Automatically generated - may contain errors
Lab products found in correlation
Hek blue htlr4, by InvivoGen (3 mentions)
Hek blue htlr3, by InvivoGen (3 mentions)
Hek blue htlr9, by InvivoGen (3 mentions)
Normocin, by InvivoGen (2 mentions)
Pen strep, by InvivoGen (2 mentions)
Bhk 21, by American Type Culture Collection (2 mentions)
L glutamine, by Corning (2 mentions)
Hek blue htlr2, by InvivoGen (2 mentions)
Hek blue htlr8, by InvivoGen (2 mentions)
Hek blue htlr2 tlr1, by InvivoGen (2 mentions)
Hek blue htlr2 tlr6, by InvivoGen (2 mentions)
Hek blue htlr5, by InvivoGen (2 mentions)
Spectramax 340pc plate reader, by Molecular Devices (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Dulbecco s modified eagle medium dmem, by Corning (1 mentions)
Spark plate reader, by Tecan (1 mentions)
Ciprofloxacin, by Corning (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Penicillin streptomycin pen strep, by Corning (1 mentions)
Minimum essential medium (mem), by Corning (1 mentions)
6 protocols using hek blue htlr7
1
Cell Culture Protocol for HEK293 and BHK-21
2
Toll-like Receptor Screening of Phages
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Toll-like receptor screening was conducted by seeding HEK-Blue hTLR2-TLR1, HEK-Blue hTLR2-TLR6, HEK-Blue hTLR2, HEK-Blue hTLR3, HEK-Blue hTLR4, HEK-Blue hTLR5, HEK-Blue hTLR7, HEK-Blue hTLR8, and HEK-Blue hTLR9 (InvivoGen) into 96-well plates. Phages OMKO1, LPS-5, PSA04, and PSA34 were added at a concentration of 1 × 1010 PFU/ml in PBS supplemented with 0.9 mM CaCl2 and 0.5 mM MgCl2 and incubated at 37°C for 24 h with 5% CO2. At the end of the incubation, NF-κB activity was monitored by the production of secreted embryonic alkaline phosphatase (SEAP) through the hydrolysis of HEK-Blue (InvivoGen). Optical density was read at 650 nm using a SpectraMax340 PC plate reader (Molecular Devices).
3
Quantifying TLR7 and RIG-I Activation
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HEK-Blue hTLR7 and HEK-Lucia RIG-I
cells, purchased from InvivoGen and maintained according to the supplier’s
guidelines in an incubator at 37 °C and 5% CO2, are
plated at 10,000 cells per well for RIG-I and 40,000 cells per well
for HTLR7 in a 96-well plate 24 h prior to transfection of all samples
using Lipofectamine 2000. 10 ng/mL 3p-hpRNA for HEK-Lucia RIG-I or
2 μg/mL R848 for HEK-Blue hTLR7 is used as a positive control
for the indicated cell line. Addition of QUANTI-Blue solution to the
HEK-Blue cell line or QUANTI-Luc solution to HEK-Lucia RIG-I cell
line following the manufacturer’s protocol gave results for
SEAP production or luciferase activity under the effect of 24 h treatment.
A Tecan Spark plate reader was used to read the absorbance at 638
nm for QUANTI-Blue after 4 h of incubation at 37 °C. For QUANTI-Luc,
the plate was read immediately for luminescence with a 100 ms reading
time.
cells, purchased from InvivoGen and maintained according to the supplier’s
guidelines in an incubator at 37 °C and 5% CO2, are
plated at 10,000 cells per well for RIG-I and 40,000 cells per well
for HTLR7 in a 96-well plate 24 h prior to transfection of all samples
using Lipofectamine 2000. 10 ng/mL 3p-hpRNA for HEK-Lucia RIG-I or
2 μg/mL R848 for HEK-Blue hTLR7 is used as a positive control
for the indicated cell line. Addition of QUANTI-Blue solution to the
HEK-Blue cell line or QUANTI-Luc solution to HEK-Lucia RIG-I cell
line following the manufacturer’s protocol gave results for
SEAP production or luciferase activity under the effect of 24 h treatment.
A Tecan Spark plate reader was used to read the absorbance at 638
nm for QUANTI-Blue after 4 h of incubation at 37 °C. For QUANTI-Luc,
the plate was read immediately for luminescence with a 100 ms reading
time.
4
Culturing HEK-Blue hTLR7 and hTLR9 Cells
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HEK-Blue hTLR7 and HEK-Blue hTLR9 cells were obtained from InvivoGen. The cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 0.2% sodium bicarbonate, 100 IU/mL penicillin, 100 μg/mL streptomycin, 2 mM l -glutamine, 30 μg/mL blasticidin, 100 μg/mL zeocin, and 100 μg/mL normocin at 37 °C in humidified air containing 5% CO2 as per the manufacturer’s instructions.
5
Toll-like Receptor Activation by Phages
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Toll-like receptor screening was conducted by seeding HEK-Blue hTLR2-TLR1, HEK-Blue hTLR2-TLR6, HEK-Blue hTLR2, HEK-Blue hTLR3, HEK-Blue hTLR4, HEK-Blue hTLR5, HEK-Blue hTLR7, HEK-Blue hTLR8, and HEK-Blue hTLR9 (InvivoGen) into 96-well plates. Phages OMKO1, LPS-5, PSA04, and PSA34 were added at a concentration of 1 x 1010 PFU/ml in PBS supplemented with 0.9 mM CaCl2 and 0.5 mM MgCl2 and incubated at 37°C for 24 h with 5% CO2. At the end of the incubation, NF-κB activity was monitored by the production of secreted embryonic alkaline phosphatase (SEAP) through the hydrolysis of HEK-Blue (InvivoGen). Optical density was read at 650 nm using a SpectraMax340 PC plate reader (Molecular Devices).
6
Culturing Cell Lines for Immunology Research
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BHK-21 (ATCC CCL-10, VA, USA) and HEK293 (ATCC CRL-1573, VA, USA) cells were cultured in Minimal Essential Media (MEM; Cellgro, NY, USA), supplemented with 10% Fetal Bovine Serum (FBS; Corning, 35-010-CV, NY, USA), 1x Penicillin/Streptomycin (Pen/Strep; Corning, 30-002-CI, NY, USA), 1x Non-essential Amino Acids (NEAA; Corning, 25-025-CI, NY, USA), and L-glutamine (Corning, 25-005-CI, NY, USA). HEK293-derived reporter cells, HEK-Blue hTLR7 (Invivogen, hkb-htlr7, CA, USA), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-017-CV, NY, USA) supplemented with 4.5 g/L glucose, 10% FBS, 1x Pen/Strep, and 1x Normocin (Invivogen, ant-nr-1, CA, USA). HEK-Blue cells were kept at low passage numbers to maintain genetic homogeneity and given selection antibiotics every other passage to maintain genomic integrity. Immortalized wild-type C57Bl/6-derived (NR-9456, BEI resources, VA, USA), and C57Bl/6 congenic MyD88-/- macrophages (NR-15633, BEI resources, VA, USA) were maintained in DMEM supplemented with 4.5 g/L glucose, 10% FBS (Corning), 2 mM L-glutamine (Corning), 1 mM Sodium Pyruvate (Corning. 25-000-CI, NY USA), and 10ug/ml ciprofloxacin (Corning, 61-277-RF).
All cells were cultured in a humidified incubator at 37°C in the presence of 5% CO2, and passaged according to standard practices for each cell line.
All cells were cultured in a humidified incubator at 37°C in the presence of 5% CO2, and passaged according to standard practices for each cell line.
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