easysep ® human cd25 positive selection cocktail kit  (STEMCELL Technologies Inc)

Journal: Journal of Clinical Medicine

Article Title: An Analysis of IL-10 , IL-17A , IL-17RA , IL-23A and IL-23R Expression and Their Correlation with Clinical Course in Patients with Psoriasis

doi: 10.3390/jcm10245834

Figure Lengend Snippet: Gene expression levels (RQ values) in lymphocytes assessed by the ΔΔCT method and percentage of samples with expression levels above and below calibrator values in the psoriatic and control groups.

Article Snippet: Following this, CD25+ T lymphocytes were positively selected from enriched CD4+ cells using the EasySep ® Human CD25 Positive Selection Cocktail kit (StemCell Technologies) and magnetic beads EasySep ® Magnetic Nanoparticles (StemCell Technologies).

Techniques: Gene Expression, Expressing, Control

Journal: Journal of immunological methods

Article Title: Isolation and expansion of human natural T regulatory cells for cellular therapy

doi: 10.1016/j.jim.2010.10.006

Figure Lengend Snippet: Purity of freshly isolated CD4+CD25++bright nTregs showing minimal contamination with other cell populations. CD4+CD25++bright cells isolated by the two step technique of negative selection of CD4+T cells followed by positive selection of CD4+CD25++bright cells using the Robosep as per protocol were stained and analyzed by flow cytometry. Upper panel shows sequential analysis of frequencies of CD3, CD4, CD25 and Foxp3 expressing cells. Lower panel shows frequencies of individual CD19, CD14, CD56 and CD8 expressing cells on (A) open gate (B) cells gated on singlets (representative of one of the two experiments).

Article Snippet: For the second step, the Robosep instrument was primed as per the manufacturer’s protocol by loading EasySep human CD25 Positive Selection Cocktail and EasySep Magnetic Nanoparticles (both from Stem Cell Technologies, Vancouver, BC, Canada) using the volume and concentration specified for the selection of CD4 + CD25 ++bright cells.

Techniques: Isolation, Selection, Staining, Flow Cytometry, Expressing

Journal: Journal of immunological methods

Article Title: Isolation and expansion of human natural T regulatory cells for cellular therapy

doi: 10.1016/j.jim.2010.10.006

Figure Lengend Snippet: Gating strategy and phenotype analysis of ex vivo expanded CD4+CD25++bright nTregs. CD4+CD25++bright cells were expanded ex vivo as per protocol and were harvested on day 19. (A) Expanded cells were stained and analyzed on open gate for viability using ViViD dye and for CD3, CD4, CD25 Foxp3, CD27, CD45RO and CD127. The CD3+CD4+cells were gated for CD25 and Foxp3. FoxP3+cells were analyzed for expression of CD127 and CD45RO+CD27+phenotype demonstrating that they were CD127 negative memory cells. (B) Cells gated on singlets and analyzed in a similar manner. The figure is representative of 4 experiments.

Article Snippet: For the second step, the Robosep instrument was primed as per the manufacturer’s protocol by loading EasySep human CD25 Positive Selection Cocktail and EasySep Magnetic Nanoparticles (both from Stem Cell Technologies, Vancouver, BC, Canada) using the volume and concentration specified for the selection of CD4 + CD25 ++bright cells.

Techniques: Ex Vivo, Staining, Expressing

Journal: Journal of immunological methods

Article Title: Isolation and expansion of human natural T regulatory cells for cellular therapy

doi: 10.1016/j.jim.2010.10.006

Figure Lengend Snippet: Ex vivo expanded nTregs exhibit potent suppressive function. Autologous CD4+CD25negative responder cells labeled with CFSE dye were analyzed for proliferation and cell division on day 4 following culture under the following conditions: (A) in medium, without stimulation, (B) after stimulation with anti-CD3/ anti-CD28 coated micro beads (positive control), (C) with anti-CD3/anti-CD28 stimulation and addition of expanded CD4+CD25++bright cells to responder cells in1:1 and 1:10 ratios and (D) with anti-CD3/anti-CD28 stimulation and addition of expanded CD4+CD25dim cells to responder cells in 1:1 and 1:10 ratios. The figure is representative of 3 experiments showing potent suppression by the CD4+CD25++bright cells at 1:1 ratio. (E) Summary data from 3 donors showing the effect of adding CD4+CD25++bright and CD4+CD25dim cells to autologous CD4+CD25negative cells stimulated with anti-CD3/anti-CD28 coated micro beads. Box plots represent mean and 95th percentiles of proliferation response (%CFSE low cells). Asterisks indicate statistical significance (**p<0.01, ***p<0.001).

Article Snippet: For the second step, the Robosep instrument was primed as per the manufacturer’s protocol by loading EasySep human CD25 Positive Selection Cocktail and EasySep Magnetic Nanoparticles (both from Stem Cell Technologies, Vancouver, BC, Canada) using the volume and concentration specified for the selection of CD4 + CD25 ++bright cells.

Techniques: Ex Vivo, Labeling, Positive Control

Journal: Journal of immunological methods

Article Title: Isolation and expansion of human natural T regulatory cells for cellular therapy

doi: 10.1016/j.jim.2010.10.006

Figure Lengend Snippet: Ex vivo expanded nTregs exhibit minimal cytokine expression following in vitro stimulation. Robosep isolated CD4+CD25++bright, CD4+CD25dim and CD4+CD25negative cells were cultured as per the expansion protocol in X-Vivo complete medium containing CD3/CD28 expander beads and rhIL2 (300 IU ml−1) and rapamycin (100 ng ml−1). Each cell population was harvested on day 19, stimulated with PMA/Ionomycin for 5 h and stained for cytokines IL17, IL2, IFNγ, TNFα and for CD107a. Each cell population was analyzed by FLOWJO using (A) an open gate (B) cells gated on singlets and analyzed in a similar manner. Plots show frequencies of cells positive for each cytokine and for CD107a in CD4+CD25++bright, CD4+CD25dim and CD4+CD25negative cells, demonstrating the very low frequencies for all measures in CD4+CD25++bright cells compared to the other two populations. (C) Summary data from 4 donors showing frequencies of IL17, IFN-γ, IL2, TNFα and CD107a expressing cells in CD4+CD25++bright cells (blue) in comparison with CD4+CD25dim (red) and CD4+CD25negative (green) cell populations. Box plots represent mean and 95th percentiles. Asterisks indicate statistical significance p b0.05 (ANOVA, Tukey post test).

Article Snippet: For the second step, the Robosep instrument was primed as per the manufacturer’s protocol by loading EasySep human CD25 Positive Selection Cocktail and EasySep Magnetic Nanoparticles (both from Stem Cell Technologies, Vancouver, BC, Canada) using the volume and concentration specified for the selection of CD4 + CD25 ++bright cells.

Techniques: Ex Vivo, Expressing, In Vitro, Isolation, Cell Culture, Staining