2) For methods # 2# 2 and 3, SB plus 0
2) For methods # 2# 2 and 3, SB plus 0.5% saponin, plus 0.5% Tween 20 (Fig. the detection of the transcription factors presence in different types of cells. Different from most cell lines, main cells are composed of Bitopertin (R enantiomer) heterogeneous populations of cells. Therefore, analysis of transcription factors by western blot analysis has major limitations because it requires prior purification of subpopulations of cells, and a large number of cells (at least 0.5106). For cells of the immune system, such separation of subpopulations can be achieved by fluorescent-activated cell sorting (FACS) based on surface markers. However, many subpopulations contain limited numbers of cells, which makes western blot analysis difficult. On the other hand, immunocytochemistry offers the advantage of allowing the identification of the transcription factors in small populations of cells and also permits to determine whether the transcription factor is present in the Bitopertin (R enantiomer) cytoplasmic or nuclear portion. This technique allows surface staining as well and identification of subpopulations, however with limited quantity of fluorochromes at the same time. In addition, you will find limitations in terms of evaluation of significant numbers of cells using this technique. The method of choice that permits quick and simultaneous identification of subpopulations expressing the transcription factors of interest in small but significant number of cells without prior purification SQSTM1 is usually circulation cytometry. The identification of subpopulations which expresses the transcription factor can be achieved by knocking in reporter genes, such as Green Fluorescence Protein (GFP) downstream of the transcription factor promoter, such as it has been achieved for Foxp3 1 and RORgammat 2. In the absence of such models, the populations expressing the transcription factor can be recognized by cell surface staining for subpopulation specific markers and intranuclear staining with antibodies for the specific transcription factor. This second goal requires permeabilization of the cellular and nuclear membranes to allow antibodies to reach the nuclear epitopes. Specifically, following surface staining, fluorochrome-labeled antibodies bound to surface markers will be fixed. Cells can be then permeabilized, which allows exposure of nuclear epitopes, followed by staining with transcription factor-specific antibodies either directly coupled with a fluorochrome or with fluorochrome-labeled secondary antibodies. At the end, samples are run on a circulation cytometer and specific subpopulations are recognized based on the surface markers together with the transcription factor of interest. This technique offers the advantage of single cell analysis, which enables the determination of both the presence of the transcription factor of interest in subpopulations of main cells, and the frequencies of the primary cell subpopulations which express Bitopertin (R enantiomer) the transcription factor without the need of the prior purification of the subpopulations. The technique was proven to render significant data after acquisition and analysis of a small number of cells, as little as 3104. In addition, the technique allows quantification of the transcription factor in subpopulations of cells by evaluation of mean fluorescence intensity (MFI). BCL11B, known also as CTIP2, is usually a sequence-specific DNA binding transcription factor 3 expressed in immune system 4C7, brain 8C12 and skin 13, 14. It has been exhibited that its expression in immune system is usually confined to T lymphocytes 4C7. It is therefore critical to establish a methodology based on circulation cytometry analysis that allows determination of various T cell subpopulations which express BCL11B. 2. Materials Mice 5C10 weeks C57BL/6 female or male mice. Mice were housed in the Albany Medical Center animal research facility and all the animal procedures were approved by the Institutional Animal Care and Use Committee. Devices and Disposables Sterile surgical devices: forceps and scissors (Roboz Surgical Instrument Co., Gaithersburg, MD). 40 m nylon cell strainers (BD Falcon, Franklin Lakes, NJ) 50 ml conical tube (BD Biosciences, Franklin Lakes, NJ) 5 ml syringes sterile Bitopertin (R enantiomer) transfer pipettes MACS multistand (Miltenyi Biotec, Bergisch Gladbach, Germany) Minimacs separation unit (Miltenyi Biotec) MS columns (Miltenyi Biotec) Sorval Story RT with cytospin system and plate holders (Thermo Scientific, Waltham, MA) Olympus BX61 Microscope (Olympus, Center Valley, PA) FACS Calibur Circulation Cytometer (Beckton Dickinson, San Jose, CA) Media and Buffers Total Medium (CM): RPMI 1640 media (Gybco/BRL, Bethesda, MD) supplemented with 10% warmth inactivated FCS (Hyclone, Logan, UT), 100 U/ml Penicillin, 100 U/ml Streptomycin answer (Gibco), 2 mM L-glutamine (Gibco), 0.05 mM beta mercaptoethanol (Sigma, St. Louis, MO) and 25 mM HEPES Red Blood Cell Lysis Buffer.