purified t helper cells (cd4)  (Hemacare Inc)

Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits the proliferation of human primary T cells. CD4+ T cells were isolated by negative selection (>97% purity), activated with immobilized CD3/CD28 Abs, and concurrently treated with the indicated concentrations of DIDOX, DOX, or vehicle control. After 24h of culture, 1 μCi of 3[H] thymidine was added to each well for an additional 24h of culture and proliferation measured using a thymidine incorporation assay. Data are normalized relative to values obtained in cultures treated with vehicle control. Data from three independent experiments are presented with error bars depicting S.E.

Article Snippet: 2.2 T cell activation and culture condition Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

Techniques: Isolation, Selection, Control, Thymidine Incorporation Assay

Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits T cell CD40L expression. Primary human CD4+ T cells were isolated by negative immunomagnetic selection (~98% purity). Cells were activated with immobilized CD3/CD28 Abs either concurrent to treatment with GPX -150 for 6h (A & B) or following an 18h DIDOX pretreatment followed by 6h CD3/CD28 activation (C & D). After activation, cells were harvested, stained with a FITC-conjugated CD40L Ab and analyzed by flow cytometry. A) Concurrent DIDOX treatment and TCR activation (6h) inhibits T cell CD40L expression, n=4. B) A representative histogram is depicted with control cultures staining 57% positive for CD40L and 50 μM DIDOX treated cultures staining 32.6% positive, iso = isotype, NT = no treatment. C) DIDOX pretreatment (18h) prior to TCR activation (6h) inhibits CD40L expression, n=5. D) A representative histogram is depicted with control cultures staining 74.6% CD40L positive and 50 μM DIDOX treated cultures staining 12.0% positive. Error bars depict S.E and asterisks indicate statistical significance from control.

Article Snippet: 2.2 T cell activation and culture condition Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

Techniques: Expressing, Isolation, Selection, Activation Assay, Staining, Flow Cytometry, Control

Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits T cell CD25 expression. Primary CD4+ T cells were isolated by negative selection (~95% purity) and pretreated with various concentrations of DIDOX or vehicle for 18h. Cells were subsequently activated with CD3/CD28 antibodies for an additional 24h, stained with a FITC-conjugated CD25 Ab, and analyzed by flow cytometry. A) Data are normalized relative to values obtained in TCR activated cultures treated with vehicle alone, n=6. Error bars depict S.E and asterisks indicate significance from baseline control. B) A representative histogram depicts CD25 staining in control cultures (NT; 73.4% CD25 positive staining) and cultures treated with 50 μM DIDOX (20.1% CD25 positive) relative to staining with an isotype control antibody (iso).

Article Snippet: 2.2 T cell activation and culture condition Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

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

Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits T cell production of TNF-α Primary CD4+ T cells were isolated by negative immunomagnetic selection (>98% purity). Cells were activated for 48h with immobilized CD3/CD28 antibodies, 40 U/ml IL-2, and treated with various concentrations of DIDOX. Cell-free conditioned media was harvested via successive centrifugations and analyzed for TNF-α by ELISA. Data from 5 representative experiments is presented. Error bars depict S.E. and asterisks indicates statistical significance from baseline control.

Article Snippet: 2.2 T cell activation and culture condition Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

Techniques: Isolation, Selection, Enzyme-linked Immunosorbent Assay, Control

Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits T cell production of IL-2. Primary CD4+ T cells were isolated by negative immunomagnetic selection (>98% purity). Cells were activated for 48h with immobilized CD3/CD28 antibodies and treated with various concentrations of DIDOX or vehicle control. Cell-free conditioned media was harvested via successive centrifugations and analyzed by ELISA. Data from 3 representative experiments is presented. Error bars depict S.E. and asterisks indicates statistical significance from baseline control.

Article Snippet: 2.2 T cell activation and culture condition Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

Techniques: Isolation, Selection, Control, Enzyme-linked Immunosorbent Assay