immunomagnetic negative magnetic selection easysep human cd4+ t cell enrichment kit  (STEMCELL Technologies Inc)

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were either left untreated (resting) or activated with plate-bound anti-CD3 and anti-CD28 antibodies. For activated cells, samples were either left untreated (no supernatant), incubated with cell-free supernatants from untreated B cells (control), or incubated with cell-free supernatants from B cells exposed to L . infantum amastigotes at a final parasite:host cell ratio of 3:1 (AMA). Seventy-two hours later, cells were analysed for cell surface expression of CD25 and CD69 by flow cytometry. Dead cells were excluded by 7-AAD staining. A) A representative dot plot is shown for each tested condition. B) Percentages of CD25 + CD69 + Tcells for studies performed with CD25 - CD4 + T cells from 3 different donors incubated with cell-free supernatants from 5 to 7 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Incubation, Control, Expressing, Flow Cytometry, Staining, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were first labeled with CFSE and treated as described in . After 5 days, cell proliferation was assessed by CFSE dilution and dead cells were excluded by 7-AAD staining. A) A representative histogram is shown for each tested condition. B) Proliferation index for experiments performed with CD25 - CD4 + T cells from 2 different donors incubated with cell-free supernatants from 6 or 7 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Labeling, Staining, Incubation, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were activated for 72 h with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of cell-free supernatants from B cells either left untreated (control) or incubated overnight with L . infantum amastigotes at a final parasite:host cell ratio of 3:1 (AMA). In some cases, cell-free supernatants from B cells incubated with parasites were treated with a soluble IL-10 receptor (1 μg/ml) before being used with activated CD25 - CD4 + T cells. During the last 5 h of incubation, cells were further stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) and Golgiplug™ was added (1 μl per 1 x 10 6 cells). Finally, cells were fixed, permeabilized and stained for intracellular TNF before being analysed by flow cytometry. Dead cells were excluded by 7-AAD staining. Results are expressed as the percentages of TNF + cells multiply by the mean fluorescence intensities (MFI). Data shown are the results from 4 different CD25 - CD4 + T-cell donors incubated with cell-free supernatants from 4 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Control, Incubation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were activated for 72 h with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of cell-free supernatants from B cells either left untreated (control) or incubated overnight with L . infantum amastigotes at a final parasite:host cell ratio of 3:1 (AMA). In some cases, cell-free supernatants from B cells incubated with parasites were treated with a soluble IL-10 receptor (1 μg/ml) before being used with activated CD25 - CD4 + T cells. During the last 5 h of incubation, cells were further stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) and Golgiplug™ was added (1 μl per 1 x 10 6 cells). Finally, cells were fixed, permeabilized and stained for intracellular IFNγ before being analysed by flow cytometry. Dead cells were excluded by 7-AAD staining. Results are expressed as the percentages of TNF-positive cells multiply by the mean fluorescence intensities (MFI). Data shown are the results from 3 different CD25 - CD4 + T-cell donors incubated with cell-free supernatants from 3 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Control, Incubation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test