Naive T-cell mediated colitis was induced in female CB.17 SCID recipient mice while CB6F1 female mice served as cell donors (Jackson Labs, Sacramento, CA). CB6F1 mice were humanely killed and spleens collected on ice. Following homogenization of spleens and RBC lysis, CD4+ cells were enriched from pooled spleen cells using a commercially available negative selection kit (Stemcell Technologies, Vancouver, BC) following the manufacturer’s protocol. These CD4+ enriched cells were stained with Alexa Flour488 conjugated anti-CD4 clone RM4-5, Alexa Flour647 conjugated anti-CD45RB clone C363-16A, and PE-conjugated anti-CD25 clone PC61 (eBioscience, San Diego, CA). CD4+ CD45RBhigh cells were sorted on a FACS Aria II (BD Biosciences, San Jose, CA). Nine recipient CB.17 SCID mice were injected intraperitoneally with 5 × 105 purified CD4+ CD45RBhigh donor lymphocytes in 200 μL of PBS whereas 9 naive animals served as a control. Fresh fecal pellets were collected at week 4 post-adoptive transfer and snap-frozen in liquid nitrogen. Samples were stored at −80°C until processed for RNA isolation.
Pe conjugated anti cd25 clone pc61
Manufactured by Thermo Fisher Scientific
PE-conjugated anti-CD25 (clone PC61) is a flow cytometry antibody reagent. It binds to the CD25 surface antigen, which is a marker of activated T cells and regulatory T cells.
Automatically generated - may contain errors
3 protocols using pe conjugated anti cd25 clone pc61
1
Adoptive Transfer of Naive T Cells Induces Colitis
2
Multiparametric Flow Cytometry Analysis of Immune Cell Subsets
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Mice were sacrificed at the indicated time points after infection and splenocytes were isolated for flow cytometry analysis. The viability of the cells was determined by Trypan blue exclusion test and only samples with viability > 98% were use for further staining. The subsets of spleen cells were defined as: CD11c + CD11b + myeloid DCs, CD11c + CD45R/B220 + plasmacytoid DCs, F4/80 + CD36 + macrophages, CD4 + T-bet + IFN-γ + T cells, and CD4 + CD25 + Foxp3 + Treg. Mature CD11c + DCs were identified by the expression of MHCII, CD86, and TLR9. Unless otherwise indicated, antibodies were purchased from BD Pharmingen. The following antibodies were used: FITC-conjugated anti-CD11c (clone HL-3), PE-conjugated anti-CD11b (clone M1/70), PerCP-conjugated anti-B220 (clone RA3-6B2), PE-conjugated anti-MHCII (clone M5/114.15.2), PEconjugated anti-CD86 (clone GL1), biotinylated anti-TLR9 (clone 5G5, purchased from Hycult biotech), PE-conjugated streptavidin (purchased from Biolegend), APC-conjugated anti-IFN-γ (XMG1.2), FITC-conjugated anti-F4/80 (clone BMB), PE-conjugated anti-CD36 (clone 72-1, purchased from eBioscience), FITC-conjugated anti-CD4 (clone GK1.5), anti-T-bet-PE (clone eBio4B10, purchased from eBioscience), PE-conjugated anti-CD25 (clone PC61), and APCconjugated anti-Foxp3 (clone FJK16s, purchased from eBioscience).
3
Quantifying Immune Cell Subsets
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Splenocytes from a portion of each group were collected to determine the relative percentage of Th1 type cells (CD4 + CD69 + ), Tregs (CD4 + CD25 + Foxp3 + ), the subsets of splenic myeloid DCs (mDC, CD11c + CD11b + ) and plasmacytoid DCs (pDC, CD-11c + B220 + ), and the expression of MHC II on DCs (CD11c + MHC II + ). Unless otherwise indicated, antibodies were purchased from BD Biosciences. When mouse cells were analysed, the following antibodies were used: FITC-conjugated anti-CD11c (clone HL-3), PE-conjugated anti-CD11b (clone M1/70), PerCP-conjugated anti-B220 (clone RA3-6B2), PE-conjugated anti-MHC II (clone M5/114.15.2, eBioscience), FITC-conjugated anti-CD69 (clone H1.2F3), FITC-conjugated anti-CD4 (clone GK1.5), PE-conjugated anti-CD25 (clone PC61), and APC-conjugated anti-Foxp3 (clone FJK16s, eBioscience). Flow cytometry was performed on a FACS Calibur (BD Biosciences, San Diego, CA, USA) and analysed using the FlowJo software (Treestar, San Carlos, CA, USA).
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