EL4 cells (Sigma-Aldrich) were grown in DMEM (Gibco). At 24 h after the cells were seeded onto a 12-well plate, the cells were treated with ligands 1–3 (from 10 mM stock in DMSO) or DMSO. After 24 h, the cells were collected and RNA was isolated using a RNeasy Plus Micro Kit (Qiagen) and reverse transcribed using the iScrip cDNA biosynthesis kit (Bio-Rad). Quantitative RT–PCR was performed to analyse mRNA levels of mouse IL17a levels using SYBR green technology (Bio-Rad) on a CFX Real-Time System (Bio-Rad). Primer sequences used for IL17a:45 (link) Fw: 5′-ctccagaaggccctcagactac-3′, Rev: 5′-ctgtgtcaatgcggagggaaagct-3′ and Gapdh: Fw: 5′-ggtggacctcatggcctaca-3′, Rev: 5′-ctctcttgctcagtgtccttgct-3′. The level of IL17a mRNA expression was normalized to that of Gapdh expression. All data are expressed as the mean±s.e.m. (n=6). Statistical analysis was performed using an one-way analysis of variance comparing against the DMSO control following Dunnett post hoc test.
El4 cells
Manufactured by Merck Group
EL4 cells are a type of mouse lymphoma cell line. They are commonly used in biomedical research as a model system for various studies, including immunology, cancer biology, and drug development. EL4 cells are well-characterized and have a defined set of characteristics that make them a valuable tool for researchers.
Automatically generated - may contain errors
Lab products found in correlation
Cfx real time system, by Bio-Rad (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Sybr green technology, by Bio-Rad (2 mentions)
A20 cells tib 208, by American Type Culture Collection (2 mentions)
Phoenix eco, by American Type Culture Collection (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Iscript advanced cdna synthesis kit, by Bio-Rad (2 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
5 protocols using el4 cells
1
Quantifying IL17a mRNA Expression in EL4 Cells
2
Generation of Murine Tumor Cell Lines
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EL4 cells (catalog number: 87020408) were purchased from Sigma. Murine CD19 was transduced into EL4 via viral transduction. Phoenix-ECO (catalog number: CRL-3214) and A20 cells (TIB-208) were purchased from ATCC. A20-MHC I and MHCII knockout lines were generated using CRISPR as described (12 (link)). Tumor cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin/streptomycin. Retroviral producing cell lines (Phoenix-ECO and 293 Glv9) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin/streptomycin. All cell lines are regularly tested for mycoplasma contamination.
3
Quantitative RT-PCR for IL-17a Expression
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EL4 cells (Sigma-Aldrich) were grown in DMEM (Gibco) with 10% FBS.
At 24 h after the cells were seeded onto a 12-well plate, the cells
were incubated with 10 μM compound (from 10 mM stock in DMSO)
or DMSO for 24 h and activated with phorbol 12-myristate 13-acetate
(PMA, 50 ng/mL; Sigma-Aldrich) and ionomycin (1 μg/mL; Sigma-Aldrich)
for 5 h. The cells were then collected, and RNA was isolated using
a RNeasy Mini Kit (Qiagen) and reverse transcribed using the iScript
Advanced cDNA Synthesis Kit (Bio-Rad). Quantitative RT-PCR was performed
to analyze mRNA levels of mouse IL-17a levels (in triplicate) using
SYBR green technology (Bio-Rad) on a CFX Real-Time System (Bio-Rad).
The following primer assays were purchased from Bio-Rad: IL-17a (qMmuCID0026592)
and Gapdh (qMmuCED0027497). The level of IL-17a mRNA expression was
normalized to that of Gapdh expression. The relative gene expression
was calculated by the 2–ΔΔCt (Livak)
method using the DMSO control as calibrator. (Data recorded in triplicate;
error shown is standard deviation from the mean; data are representative
of >3 repeated experiments).
At 24 h after the cells were seeded onto a 12-well plate, the cells
were incubated with 10 μM compound (from 10 mM stock in DMSO)
or DMSO for 24 h and activated with phorbol 12-myristate 13-acetate
(PMA, 50 ng/mL; Sigma-Aldrich) and ionomycin (1 μg/mL; Sigma-Aldrich)
for 5 h. The cells were then collected, and RNA was isolated using
a RNeasy Mini Kit (Qiagen) and reverse transcribed using the iScript
Advanced cDNA Synthesis Kit (Bio-Rad). Quantitative RT-PCR was performed
to analyze mRNA levels of mouse IL-17a levels (in triplicate) using
SYBR green technology (Bio-Rad) on a CFX Real-Time System (Bio-Rad).
The following primer assays were purchased from Bio-Rad: IL-17a (qMmuCID0026592)
and Gapdh (qMmuCED0027497). The level of IL-17a mRNA expression was
normalized to that of Gapdh expression. The relative gene expression
was calculated by the 2–ΔΔCt (Livak)
method using the DMSO control as calibrator. (Data recorded in triplicate;
error shown is standard deviation from the mean; data are representative
of >3 repeated experiments).
4
Generation of Murine Tumor Cell Lines
5
Quantifying IL-17A mRNA Levels in EL4 Cells
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+ Expand
This assay was conducted using EL4 cells (Sigma-Aldrich), cultured in DMEM (Gibco) with 10% FBS. After seeding the cells onto a 12-well plate, they were incubated in the presence of 10 μM compound (n = 3, prepared from DMSO stock solutions) or DMSO (vehicle control) for 24 h. Subsequently, the cells were activated with phorbol 12-myristate 13-acetate (PMA, 50 ng mL−1; Sigma-Aldrich) and ionomycin (1 μg mL−1; Sigma-Aldrich) for 5 h. Following activation, the cells were collected, and RNA was isolated using a RNeasy Mini Kit (Qiagen). Reverse transcription was performed using the iScript Advanced cDNA Synthesis Kit (Bio-Rad). Quantitative RT-PCR was carried out to assess mRNA levels of mouse IL-17A (n = 3) utilizing SYBR green technology (Bio-Rad) on a CFX Real-Time System (Bio-Rad). Normalization of IL-17A mRNA expression to Gapdh expression was performed, and the relative gene expression was calculated using the 2−ΔΔCt (Livak) method with the DMSO control as the calibrator. This assay was performed in a single run (n = 3). The statistical significance levels were denoted as follows: ***p < 0.001, ****p < 0.0001, and ‘ns’ indicating non-significance in comparison to the vehicle control.
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