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rosetteseptm human cd3 + depletion kit (STEMCELL Technologies Inc)

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STEMCELL Technologies Inc rosetteseptm human cd3 + depletion kit

A, Magnetic bead purified CD4+ T cells were stained with CFSE (2.5μM) and stimulated with plate-bound <t>anti-CD3</t> antibody (2μg/ml) for three days together with Pam3CSK4 (4μg/ml), R-848 (1μg/ml), or ORNs (1μM) formulated with 12.5 μg/ml DOTAP. The proliferation of T cells was measured as the dilution of CFSE. One representative out of six experiments is shown. B, Isolated T cells were stimulated like in (A) and proliferation was calculated as the dilution of CFSE. Concentration of ORNs (R-0006, R-0002, R-1263) was 0.125μM, prepared with 1.55μg/ml DOTAP. Data shown represent mean ± SEM of five donors. C, Supernatants of the T cell cultures were harvested and IFN-γ production was measured in an ELISA. For stimulation without <t>anti-CD3</t> antibody, 100 U/ml IL-2 were used. Data shown represent mean ± SEM of six donors. Significance of differences was determined by Student’s t-test.

Rosetteseptm Human Cd3 + Depletion Kit, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1 "

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

Journal:

doi: 10.1016/j.imbio.2010.03.011

A, Magnetic bead purified CD4+ T cells were stained with CFSE (2.5μM) and stimulated with plate-bound anti-CD3 antibody (2μg/ml) for three days together with Pam3CSK4 (4μg/ml), R-848 (1μg/ml), or ORNs (1μM) formulated with 12.5 μg/ml DOTAP. The proliferation of T cells was measured as the dilution of CFSE. One representative out of six experiments is shown. B, Isolated T cells were stimulated like in (A) and proliferation was calculated as the dilution of CFSE. Concentration of ORNs (R-0006, R-0002, R-1263) was 0.125μM, prepared with 1.55μg/ml DOTAP. Data shown represent mean ± SEM of five donors. C, Supernatants of the T cell cultures were harvested and IFN-γ production was measured in an ELISA. For stimulation without anti-CD3 antibody, 100 U/ml IL-2 were used. Data shown represent mean ± SEM of six donors. Significance of differences was determined by Student’s t-test.

Figure Legend Snippet: A, Magnetic bead purified CD4+ T cells were stained with CFSE (2.5μM) and stimulated with plate-bound anti-CD3 antibody (2μg/ml) for three days together with Pam3CSK4 (4μg/ml), R-848 (1μg/ml), or ORNs (1μM) formulated with 12.5 μg/ml DOTAP. The proliferation of T cells was measured as the dilution of CFSE. One representative out of six experiments is shown. B, Isolated T cells were stimulated like in (A) and proliferation was calculated as the dilution of CFSE. Concentration of ORNs (R-0006, R-0002, R-1263) was 0.125μM, prepared with 1.55μg/ml DOTAP. Data shown represent mean ± SEM of five donors. C, Supernatants of the T cell cultures were harvested and IFN-γ production was measured in an ELISA. For stimulation without anti-CD3 antibody, 100 U/ml IL-2 were used. Data shown represent mean ± SEM of six donors. Significance of differences was determined by Student’s t-test.

Techniques Used: Purification, Staining, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay

A, PBMCs were isolated from different donors, stained with CFSE (2.5μM) and stimulated with anti-CD3 antibody (2μg/ml) and the indicated ligands on 96-well plates for three days. ORNs were formulated with DOTAP (4μM ORN with 50μg/ml DOTAP). ODNs were used in the absence of DOTAP. For analysis PBMCs were stained with mAb to CD4, CD8 (B) and CD19 (C), and the proliferation of the different cell types was measured as dilution of CFSE fluorescence. Data shown represent mean ± SEM of eleven (A, B) and 3 donors (C).

Figure Legend Snippet: A, PBMCs were isolated from different donors, stained with CFSE (2.5μM) and stimulated with anti-CD3 antibody (2μg/ml) and the indicated ligands on 96-well plates for three days. ORNs were formulated with DOTAP (4μM ORN with 50μg/ml DOTAP). ODNs were used in the absence of DOTAP. For analysis PBMCs were stained with mAb to CD4, CD8 (B) and CD19 (C), and the proliferation of the different cell types was measured as dilution of CFSE fluorescence. Data shown represent mean ± SEM of eleven (A, B) and 3 donors (C).

Techniques Used: Isolation, Staining, Fluorescence

A, PBMCs were stimulated with 2.5μg/ml TT for six days together with the indicated TLR agonists. ORN R-0006 and R-1263 were formulated with DOTAP (1μM ORN with 12.5μg/ml DOTAP). The recall antigen stimulation of CD4+ T cells within PBMCs was measured as the dilution of CFSE. Data shown are mean ± SEM of six donors. B, PBMCs were stimulated with 2μg/ml SEB for three days and proliferation of CD4+ and CD8+ T cells was measured. Data shown represent mean ± SEM of six donors. C, T cells from one donor and CD3 depleted APCs from another donor were isolated and mixed in a 5:1 ratio (T cells:APCs in a MLR) for three days. For inhibition of IDO function, two different concentrations of 1-MT (100μM and 500μM) were added to the cultures. Proliferation of T cells in allogenic MLR was measured as the dilution of CFSE. One experiment out of three is shown. D, PBMCs were stimulated with the indicated ligands (1.3μM CpG C-Class ODN 2395 or ORN R-0006 with DOTAP 16.6μg/ml) or 20ng/ml IFN-γ for 24h. Plates were washed and RNA from plate bound monocytes was prepared. IDO mRNA levels relative to 1000 copies cyclophilin B are depicted. Data shown are mean ± SEM of three donors

Figure Legend Snippet: A, PBMCs were stimulated with 2.5μg/ml TT for six days together with the indicated TLR agonists. ORN R-0006 and R-1263 were formulated with DOTAP (1μM ORN with 12.5μg/ml DOTAP). The recall antigen stimulation of CD4+ T cells within PBMCs was measured as the dilution of CFSE. Data shown are mean ± SEM of six donors. B, PBMCs were stimulated with 2μg/ml SEB for three days and proliferation of CD4+ and CD8+ T cells was measured. Data shown represent mean ± SEM of six donors. C, T cells from one donor and CD3 depleted APCs from another donor were isolated and mixed in a 5:1 ratio (T cells:APCs in a MLR) for three days. For inhibition of IDO function, two different concentrations of 1-MT (100μM and 500μM) were added to the cultures. Proliferation of T cells in allogenic MLR was measured as the dilution of CFSE. One experiment out of three is shown. D, PBMCs were stimulated with the indicated ligands (1.3μM CpG C-Class ODN 2395 or ORN R-0006 with DOTAP 16.6μg/ml) or 20ng/ml IFN-γ for 24h. Plates were washed and RNA from plate bound monocytes was prepared. IDO mRNA levels relative to 1000 copies cyclophilin B are depicted. Data shown are mean ± SEM of three donors

Techniques Used: Isolation, Inhibition

A, PBMCs were stimulated with the different ORNs (4μM with 50μg/ml DOTAP) for 24h and stained with AnnexinV and PI together with anti-CD3 Ab. Percentage of the AnnexinV−, PI− population within the whole PBMCs or within the pre-gated CD3+ T cell population (B) is indicated. Data shown represent mean ± SEM of three donors.

Figure Legend Snippet: A, PBMCs were stimulated with the different ORNs (4μM with 50μg/ml DOTAP) for 24h and stained with AnnexinV and PI together with anti-CD3 Ab. Percentage of the AnnexinV−, PI− population within the whole PBMCs or within the pre-gated CD3+ T cell population (B) is indicated. Data shown represent mean ± SEM of three donors.

Techniques Used: Staining

A, Schematic overview of the transwell assay system. Flat bottom 96-well plates were coated with anti-CD3 Ab (2μg/ml) and T cells were cultured on these plates. In the transwell chamber T cell depleted PBMCs were stimulated. T cells and T cell depleted PBMCs from one donor were isolated and mixed either directly together (5×105 T cells with 1×105 T cell depleted PBMCs) for the control plate (C), or were stimulated in different chambers in the transwell system (B). Before mixing, T cells were stained with 2.5μM CFSE. Cells were stimulated for three days with the indicated ligands. ORNs were formulated with DOTAP (4μM with 50μg/ml DOTAP). As control T cells were stimulated with anti-CD3 Ab together with 100 U/ml IL-2. Data shown represent mean ± SEM of 4 donors.

Figure Legend Snippet: A, Schematic overview of the transwell assay system. Flat bottom 96-well plates were coated with anti-CD3 Ab (2μg/ml) and T cells were cultured on these plates. In the transwell chamber T cell depleted PBMCs were stimulated. T cells and T cell depleted PBMCs from one donor were isolated and mixed either directly together (5×105 T cells with 1×105 T cell depleted PBMCs) for the control plate (C), or were stimulated in different chambers in the transwell system (B). Before mixing, T cells were stained with 2.5μM CFSE. Cells were stimulated for three days with the indicated ligands. ORNs were formulated with DOTAP (4μM with 50μg/ml DOTAP). As control T cells were stimulated with anti-CD3 Ab together with 100 U/ml IL-2. Data shown represent mean ± SEM of 4 donors.

Techniques Used: Transwell Assay, Cell Culture, Isolation, Control, Staining

A, PBMCs were depleted of CD14+ monocytes by magnetic bead separation. Depleted PBMCs or non-depleted PBMCs, as control, were stained with CFSE (2.5μM) and stimulated for three days with anti-CD3 antibody (2μg/ml) and the indicated TLR ligands. ORNs were formulated with DOTAP (2μM ORN with 25μg/ml DOTAP). Proliferation of CD4+ and CD8+ T cells was measured after three days of culture. Data shown are mean ± SEM of six donors. B, PBMCs were stimulated for 24h with the indicated ligands with 0.1μM ORNs formulated with 1.25μg/ml DOTAP or 1μM ORN formulated with 12.5μg/ml DOTAP. PBMCs were stained with a cocktail of anti-CD19, anti-CD56, anti-CD3 antibodies together with anti-PD-L1 antibody. Monocytes (as CD56−CD3−CD19− population) were analyzed for PD-L1 expression after 24h. Data shown represent mean ± SEM of six donors (two independent experiments). Significance of differences was determined by Student’s t-test. C, PBMCs were stained with CFSE (2.5μM) and stimulated for three days with anti-CD3 antibody (2μg/ml) and the indicated TLR ligands. ORNs were formulated with DOTAP (2μM ORN with 25μg/ml DOTAP). Proliferation of CD4+ and CD8+ T cells was measured after three days of culture. Data shown are mean ± SEM of three donors. D, Splenocytes from C57BL/6 wt mice were isolated and stained with CFSE (2.5μM), and stimulated with the indicated TLR ligands. ORNs were formulated with DOTAP (4μM ORN with 50μg/ml DOTAP). Data shown represent mean ± SEM of four wt C57BL/6 mice.

Figure Legend Snippet: A, PBMCs were depleted of CD14+ monocytes by magnetic bead separation. Depleted PBMCs or non-depleted PBMCs, as control, were stained with CFSE (2.5μM) and stimulated for three days with anti-CD3 antibody (2μg/ml) and the indicated TLR ligands. ORNs were formulated with DOTAP (2μM ORN with 25μg/ml DOTAP). Proliferation of CD4+ and CD8+ T cells was measured after three days of culture. Data shown are mean ± SEM of six donors. B, PBMCs were stimulated for 24h with the indicated ligands with 0.1μM ORNs formulated with 1.25μg/ml DOTAP or 1μM ORN formulated with 12.5μg/ml DOTAP. PBMCs were stained with a cocktail of anti-CD19, anti-CD56, anti-CD3 antibodies together with anti-PD-L1 antibody. Monocytes (as CD56−CD3−CD19− population) were analyzed for PD-L1 expression after 24h. Data shown represent mean ± SEM of six donors (two independent experiments). Significance of differences was determined by Student’s t-test. C, PBMCs were stained with CFSE (2.5μM) and stimulated for three days with anti-CD3 antibody (2μg/ml) and the indicated TLR ligands. ORNs were formulated with DOTAP (2μM ORN with 25μg/ml DOTAP). Proliferation of CD4+ and CD8+ T cells was measured after three days of culture. Data shown are mean ± SEM of three donors. D, Splenocytes from C57BL/6 wt mice were isolated and stained with CFSE (2.5μM), and stimulated with the indicated TLR ligands. ORNs were formulated with DOTAP (4μM ORN with 50μg/ml DOTAP). Data shown represent mean ± SEM of four wt C57BL/6 mice.

Techniques Used: Control, Staining, Expressing, Isolation

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Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, Magnetic bead purified CD4+ T cells were stained with CFSE (2.5μM) and stimulated with plate-bound anti-CD3 antibody (2μg/ml) for three days together with Pam3CSK4 (4μg/ml), R-848 (1μg/ml), or ORNs (1μM) formulated with 12.5 μg/ml DOTAP. The proliferation of T cells was measured as the dilution of CFSE. One representative out of six experiments is shown. B, Isolated T cells were stimulated like in (A) and proliferation was calculated as the dilution of CFSE. Concentration of ORNs (R-0006, R-0002, R-1263) was 0.125μM, prepared with 1.55μg/ml DOTAP. Data shown represent mean ± SEM of five donors. C, Supernatants of the T cell cultures were harvested and IFN-γ production was measured in an ELISA. For stimulation without anti-CD3 antibody, 100 U/ml IL-2 were used. Data shown represent mean ± SEM of six donors. Significance of differences was determined by Student’s t-test.

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Purification, Staining, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, PBMCs were isolated from different donors, stained with CFSE (2.5μM) and stimulated with anti-CD3 antibody (2μg/ml) and the indicated ligands on 96-well plates for three days. ORNs were formulated with DOTAP (4μM ORN with 50μg/ml DOTAP). ODNs were used in the absence of DOTAP. For analysis PBMCs were stained with mAb to CD4, CD8 (B) and CD19 (C), and the proliferation of the different cell types was measured as dilution of CFSE fluorescence. Data shown represent mean ± SEM of eleven (A, B) and 3 donors (C).

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Isolation, Staining, Fluorescence

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, PBMCs were stimulated with 2.5μg/ml TT for six days together with the indicated TLR agonists. ORN R-0006 and R-1263 were formulated with DOTAP (1μM ORN with 12.5μg/ml DOTAP). The recall antigen stimulation of CD4+ T cells within PBMCs was measured as the dilution of CFSE. Data shown are mean ± SEM of six donors. B, PBMCs were stimulated with 2μg/ml SEB for three days and proliferation of CD4+ and CD8+ T cells was measured. Data shown represent mean ± SEM of six donors. C, T cells from one donor and CD3 depleted APCs from another donor were isolated and mixed in a 5:1 ratio (T cells:APCs in a MLR) for three days. For inhibition of IDO function, two different concentrations of 1-MT (100μM and 500μM) were added to the cultures. Proliferation of T cells in allogenic MLR was measured as the dilution of CFSE. One experiment out of three is shown. D, PBMCs were stimulated with the indicated ligands (1.3μM CpG C-Class ODN 2395 or ORN R-0006 with DOTAP 16.6μg/ml) or 20ng/ml IFN-γ for 24h. Plates were washed and RNA from plate bound monocytes was prepared. IDO mRNA levels relative to 1000 copies cyclophilin B are depicted. Data shown are mean ± SEM of three donors

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Isolation, Inhibition

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, PBMCs were stimulated with the different ORNs (4μM with 50μg/ml DOTAP) for 24h and stained with AnnexinV and PI together with anti-CD3 Ab. Percentage of the AnnexinV−, PI− population within the whole PBMCs or within the pre-gated CD3+ T cell population (B) is indicated. Data shown represent mean ± SEM of three donors.

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Staining

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, Schematic overview of the transwell assay system. Flat bottom 96-well plates were coated with anti-CD3 Ab (2μg/ml) and T cells were cultured on these plates. In the transwell chamber T cell depleted PBMCs were stimulated. T cells and T cell depleted PBMCs from one donor were isolated and mixed either directly together (5×105 T cells with 1×105 T cell depleted PBMCs) for the control plate (C), or were stimulated in different chambers in the transwell system (B). Before mixing, T cells were stained with 2.5μM CFSE. Cells were stimulated for three days with the indicated ligands. ORNs were formulated with DOTAP (4μM with 50μg/ml DOTAP). As control T cells were stimulated with anti-CD3 Ab together with 100 U/ml IL-2. Data shown represent mean ± SEM of 4 donors.

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Transwell Assay, Cell Culture, Isolation, Control, Staining

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, PBMCs were depleted of CD14+ monocytes by magnetic bead separation. Depleted PBMCs or non-depleted PBMCs, as control, were stained with CFSE (2.5μM) and stimulated for three days with anti-CD3 antibody (2μg/ml) and the indicated TLR ligands. ORNs were formulated with DOTAP (2μM ORN with 25μg/ml DOTAP). Proliferation of CD4+ and CD8+ T cells was measured after three days of culture. Data shown are mean ± SEM of six donors. B, PBMCs were stimulated for 24h with the indicated ligands with 0.1μM ORNs formulated with 1.25μg/ml DOTAP or 1μM ORN formulated with 12.5μg/ml DOTAP. PBMCs were stained with a cocktail of anti-CD19, anti-CD56, anti-CD3 antibodies together with anti-PD-L1 antibody. Monocytes (as CD56−CD3−CD19− population) were analyzed for PD-L1 expression after 24h. Data shown represent mean ± SEM of six donors (two independent experiments). Significance of differences was determined by Student’s t-test. C, PBMCs were stained with CFSE (2.5μM) and stimulated for three days with anti-CD3 antibody (2μg/ml) and the indicated TLR ligands. ORNs were formulated with DOTAP (2μM ORN with 25μg/ml DOTAP). Proliferation of CD4+ and CD8+ T cells was measured after three days of culture. Data shown are mean ± SEM of three donors. D, Splenocytes from C57BL/6 wt mice were isolated and stained with CFSE (2.5μM), and stimulated with the indicated TLR ligands. ORNs were formulated with DOTAP (4μM ORN with 50μg/ml DOTAP). Data shown represent mean ± SEM of four wt C57BL/6 mice.

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Control, Staining, Expressing, Isolation

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