TEC protein, human

TECs were isolated by flow cytometry using the protocol of Gray et al [10 (link)]. Single cell suspensions of thymocytes and splenic cells were stained with the fluorochrome-conjugated antibodies as described [11 (link)]. For intracellular staining, the cells were first permeabilized with a BD Cytofix/Cytoperm solution for 20 minutes at 4°C. Direct or indirect staining of fluorochrome-conjugated antibodies included: CD4, CD8, CD25, CD44, CD62L, c-kit, IL-7Rα, BP-1, CD45, I-A, H-2K^b, CD45.1, Ki67, CD69, EpCAM1, IL-2, IFN-γ, TNFα, Bcl-2, and a panel of TCR Vβ clonotypes (BioLegend, BD Biosciences, San Jose, CA, or eBioscience, San Diego, CA), as well as Bcl-xL (Cell Signaling Technology, Inc., Danvers, MA). ETPs were identified by phenotypic analysis (Lineage^- c-kit⁺ IL-7Rα^- CD44⁺CD25^-) as described [8 (link),11 (link)]. An antibody cocktail, composed of antibodies against TER-119, B220, CD19, IgM, Gr-1, CD11b, CD11c, NK1.1, TCRβ, CD3e, and CD8α, was used to identify lineage negative cells. Annexin V and terminal deoxynucleotidyltrasferase dUTP nick end labeling (TUNEL, APO-DIRECT) apoptosis detections kits were purchased from BD Biosciences. The samples were analyzed on a FACSCalibur or LSR II flow cytometer (BD Biosciences).

The synthesis of silica gels with various surface modifications was performed as adapted from Wei et al. [33 (link)]. A total of 6.01 g of silica (9.35 wt% of the final gel) was produced as follows: 20.84 g of TEOS was mixed with 8.78 mL of ethanol, as well as 8.77 mL of an ethanol/37% hydrochloric acid solution (438.19 mL/105 µL). Then, 1.81 mL of DIW was used to form a sol. This solution was stirred for 90 min. Afterwards, 29.23 mL of ethanol and 4.69 mL of a DIW/25% ammonium hydroxide solution (168 g/1 g) were added, and the solution was stirred for 45 min. This solution was left to gel in custom-made Teflon molds, where one gap was 1.5 cm by 1 cm by 0.6 cm. Aging of the cuboid samples was conducted for 24 h at 50 °C.
Afterwards, a solvent exchange was performed with an excess of ethanol, mixtures of ethanol and hexane (25 vol%/75 vol%; 50 vol%/50 vol%; 75 vol%/25 vol%), and four times pure hexane for 24 h, each at room temperature. The surface modification was done under equal conditions for the different silylation agents, using subsequently a 3 vol% and 6 vol% solution of silylation agent in hexane, repeating each step twice, for a total of four individual steps. The gels were modified using TMCS 108.64 gmol⁻¹ (denoted as TM), TECS 150.72 gmol⁻¹ (denoted as TE), and HMDS 161.39 gmol⁻¹ (denoted as HM), respectively. This translates to molarity of 0.236 M, 0.179 M, and 0.144 M of TMCS, TECS, and HMDS hexane mixtures for the 3 vol% solutions, as well as 0.473 M, 0.357 M, and 0.288 M for the 6 vol% solutions, respectively. Residues of the surface modification were rinsed with hexane four individual times at roughly 24 h intervals. Some samples were left unmodified (denoted as UN) and used as references. Finally, the samples were left to dry at moderate evaporation speeds of up to three days.