Tnaive (TN)(CD4+RFP−CD44−), TM (CD4+RFP−CD44+), Helios− Treg (CD4+RFP+GFP−) and Helios+ Treg (CD4+RFP+GFP+) cells from pooled spleen and LN were obtained by cell sort. RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen, Germantown, MD) followed by cDNA synthesis (Invitrogen SuperScript III First-Strand Synthesis SuperMix for qRT-PCR) (Waltham MA). RT-PCR was performed in triplicate using Applied Biosystems TaqMan Universal PCR Master Mix and run on the Applied Biosystems QuantStudio 7 Flex machine (Waltham, MA). dCt was calculated by subtracting the average value for the 18s rRNA (Applied Biosystems) from the average value for each of the other genes and changed from logarithmic to linear form using the formula 2^(-dCt). The following TaqMan Gene Expression Assays were obtained from Applied Biosystems: Tgfbr (Mm00803538_m1) Satb1 (Mm01268940_m1) Vipr1 (Mm00449214_m1) Dapl1 (Mm01271524_m1) Pde3b (Mm00691635_m1) Amigo2 (Mm00662105_s1) Gbp2b (Mm00657086) Rorc (Mm01261022_m1) Itgb8 (Mm00623991_m1) Ikzf4 (Mm01133256_m1) Penk (Mm01212875_m1) and 18s rRNA.
Pde3b
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pde3b is a laboratory equipment product designed for use in scientific research and analysis. It serves as a tool for the measurement and analysis of specific biological or chemical processes. The core function of Pde3b is to facilitate accurate and reliable data collection in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
18s rrna, by Thermo Fisher Scientific (1 mentions)
Quantstudio 7 flex machine, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis supermix for qrt pcr, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
2 protocols using pde3b
1
Sorting T Cell Subsets and Gene Expression
2
Quantitative RT-PCR Analysis of Tolerized T Cells
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Total cellular RNA was isolated from purified cell subsets from saline-treated or pCons-tolerized BWF1 mice with TRIzol (Invitrogen, Carlsbad, CA, USA) as per manufacturer’s protocols. One-step-real time PCR was analyzed as described earlier (3 (link), 5 (link), 26 (link), 29 (link)). Each experimental group consists of the pooled spleen cells of 3–4 mice from each group, naïve CD8+ T cells or tolerized CD8+ T cells. One-step RT-PCR was performed (Applied Biosystems, Foster City, CA, USA) using 100 ng of total RNA. Quantitative real-time reverse transcription was performed using TaqMan technology on an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). Primers and probes of regulator of G protein signaling genes (RGS2, RGS16, and RGS17), glutamic pyruvate transaminase 2 (GPT2), BAX (Bcl-2-associated X protein), programmed cell death-1 (PD1), growth arrest and DNA damage inducible 45 beta (GADD45β), and phosphodiesterase 3b (PDE3b), and GAPDH were obtained from Applied Biosystems (Foster City, CA, USA). The other oligonucleotide sequences used for the primers and TaqMan probes (Applied Biosystem, Foster City, CA) are described (3 (link), 5 (link), 26 (link), 29 (link)). GAPDH was used as an endogenous control in each experimental set.
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