SEARCH

SEARCH BY CITATION

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22035_sm_SuppInfo.doc135KSupporting Information
MIFlowCyt_Item_Location.doc45KSupporting Information
CYTO_22035_sm_SuppInfoFig1A.tif337KOnline Fig.1 Reagent titrations. Reagents were titrated on healthy donor PBMC by serial 2-fold dilutions. A Plots represent concatenated .fcs files, with fluorescent intensity on the y-axis and Ab concentration on the x-axis. Titers used in OMIP-xxx are highlighted in red. B Titration curves of the plots shown in A with the ratio of the median fluorescence intensity (MFI; positive MFI/negative MFI) on the y-axis and Ab concentration on the y-axis. Titers used in OMIP-xxx are represented as open symbols. Ab concentrations are “μl Ab / 100 μl buffer” for all Abs except IL-4, IL-10, and IFN-γ, which are “μg Ab / ml”.
CYTO_22035_sm_SuppInfoFig1B.tif100KOnline Fig.1 Reagent titrations. Reagents were titrated on healthy donor PBMC by serial 2-fold dilutions. A Plots represent concatenated .fcs files, with fluorescent intensity on the y-axis and Ab concentration on the x-axis. Titers used in OMIP-xxx are highlighted in red. B Titration curves of the plots shown in A with the ratio of the median fluorescence intensity (MFI; positive MFI/negative MFI) on the y-axis and Ab concentration on the y-axis. Titers used in OMIP-xxx are represented as open symbols. Ab concentrations are “μl Ab / 100 μl buffer” for all Abs except IL-4, IL-10, and IFN-γ, which are “μg Ab / ml”.
CYTO_22035_sm_SuppInfoFig2A.tif61KOnline Fig.2 Total CD4+ T cells were isolated from healthy donors and stained for Th2 cytokines. A CD4+ T cells were cultured under Th2-biasing conditions using plate-bound anti-CD3 (10 μg/ml, clone OKT3), soluble anti-CD28 (2 μg/ml, clone CD28.2), IL-2 (40 IU/ml), IL-4 (12.5 ng/ml), anti-IFNγ (5 μg/ml, clone B27), and anti-IL-10 (5 μg/ml, clone JES3-9D7). After 4 days, cells were transferred to new plates and expanded for 3 days in the above media without anti-CD3 or anti-CD28 antibodies. Cells were subsequently cultured in the presence of PMA (20 ng/ml) and Ionomycin (1 μM) (left plot) or its absence (right plot) for 4 hrs in the presence of BFA and monensin. Surface-bound IL-4 was blocked with unconjugated anti-IL-4 Ab (clone 8D4-8) prior to permeabilization, and was followed by staining with Ax488-conjugated 8D4-8. B CD4+ T cells were activated with plate-bound anti-CD3, soluble anti-CD28, and IL-2 (at concentrations used in A) for 4 days. Cells were subsequently cultured in the presence of PMA (20 ng/ml) and Ionomycin (1 μM) (left plot) or its absence (right plot) for 6 hrs in the presence of BFA and monensin. Data presented are not those in Online Fig.1. Titers used in OMIP-xxx are highlighted in red.
CYTO_22035_sm_SuppInfoFig2B.tif49KOnline Fig.2 Total CD4+ T cells were isolated from healthy donors and stained for Th2 cytokines. A CD4+ T cells were cultured under Th2-biasing conditions using plate-bound anti-CD3 (10 μg/ml, clone OKT3), soluble anti-CD28 (2 μg/ml, clone CD28.2), IL-2 (40 IU/ml), IL-4 (12.5 ng/ml), anti-IFNγ (5 μg/ml, clone B27), and anti-IL-10 (5 μg/ml, clone JES3-9D7). After 4 days, cells were transferred to new plates and expanded for 3 days in the above media without anti-CD3 or anti-CD28 antibodies. Cells were subsequently cultured in the presence of PMA (20 ng/ml) and Ionomycin (1 μM) (left plot) or its absence (right plot) for 4 hrs in the presence of BFA and monensin. Surface-bound IL-4 was blocked with unconjugated anti-IL-4 Ab (clone 8D4-8) prior to permeabilization, and was followed by staining with Ax488-conjugated 8D4-8. B CD4+ T cells were activated with plate-bound anti-CD3, soluble anti-CD28, and IL-2 (at concentrations used in A) for 4 days. Cells were subsequently cultured in the presence of PMA (20 ng/ml) and Ionomycin (1 μM) (left plot) or its absence (right plot) for 6 hrs in the presence of BFA and monensin. Data presented are not those in Online Fig.1. Titers used in OMIP-xxx are highlighted in red.
CYTO_22035_sm_SuppInfoFig3A.tif33KOnline Fig.3 Cytokine response from a T cell line from the blood of a melanoma patient stimulated with PMA and Ionomycin or unstimulated. To emphasize the Th2 response, IL-10 is displayed on the y-axis and IL-4 on the x-axis. A Shown are the CD4+ T cells stimulated (left panel) and unstimulated (right panel). B Shown are the CD8+ T cells stimulated (left panel) and unstimulated (right panel). In all panels, the cells are gated as described in Figure 1.
CYTO_22035_sm_SuppInfoFig3B.tif30KOnline Fig.3 Cytokine response from a T cell line from the blood of a melanoma patient stimulated with PMA and Ionomycin or unstimulated. To emphasize the Th2 response, IL-10 is displayed on the y-axis and IL-4 on the x-axis. A Shown are the CD4+ T cells stimulated (left panel) and unstimulated (right panel). B Shown are the CD8+ T cells stimulated (left panel) and unstimulated (right panel). In all panels, the cells are gated as described in Figure 1.
CYTO_22035_sm_SuppInfoFig4A.tif246KOnline Fig.4 Cytokine response from Hut78, a cutaneous CD4+ T cell lymphoma cell line, following various in vitro stimulations. A Hut78 was stimulated with PMA (60 ng/ml) for 6 hours (upper panel) or unstimulated (lower panel). B Hut78 was stimulated with plate-bound anti-CD3 (5 μg/ml, clone OKT3), plate-bound anti-CD3 + 10 ng/ml IL-2, or soluble anti-CD3 (1 μg/ml, clone OKT3) + soluble anti-CD28 (1 μg/ml, clone CD28.2), or were unstimulated. The gates from the unstimulated sample were copied onto the stimulated samples. Shown are the IFN-γ vs IL-4 and the IL-10 vs IL-2 responses as overlays.
CYTO_22035_sm_SuppInfoFig4B.tif81KOnline Fig.4 Cytokine response from Hut78, a cutaneous CD4+ T cell lymphoma cell line, following various in vitro stimulations. A Hut78 was stimulated with PMA (60 ng/ml) for 6 hours (upper panel) or unstimulated (lower panel). B Hut78 was stimulated with plate-bound anti-CD3 (5 μg/ml, clone OKT3), plate-bound anti-CD3 + 10 ng/ml IL-2, or soluble anti-CD3 (1 μg/ml, clone OKT3) + soluble anti-CD28 (1 μg/ml, clone CD28.2), or were unstimulated. The gates from the unstimulated sample were copied onto the stimulated samples. Shown are the IFN-γ vs IL-4 and the IL-10 vs IL-2 responses as overlays.
CYTO_22035_sm_SuppInfoFig5.tif150KOnline Fig.5 Selection of CD4 reagent. Healthy donor PBMC were stimulated with PMA + ionomycin for 4 hrs. The cells were fixed and permeabilized followed by staining with anti-CD4 antibody conjugates. Shown are CD4+ T cells from the live lymphocyte gate. The antibody conjugate (APC-Cy7) selected for use in the OMIP is indicated with an asterisk. Clone RPA-T4 was conjugated to FITC, PE-Cy7, APC-H7, or APC-Cy7; clone L200 was conjugated to PerCP; clone S3.5 was conjugated to Pacific Orange or QD605; clone OKT4 was conjugated to A×700.
CYTO_22035_sm_SuppInfoFig6A.tif262KOnline Fig.6 Staining and biological controls. A T cell line from a human melanoma tumor was stimulated with allogeneic tumor. The intracellular cytokines were blocked using purified Ab (IL-4, IL-10, TNF, IFN-γ) prior to staining with fluorochrome-conjugated Ab or the fluorochrome-conjugated Ab was blocked with recombinant human cytokine (IL-2) prior to addition to the cells (lower panels). The cytokine+ gates from the blocked sample are copied onto the unblocked sample for determination of the frequency of cytokine+ cells induced by the stimulation (upper panels). Shown are the CD4+ T cells from the live lymphocyte gate. B T cell line with known IFN-γ response to autologous tumor included as a biological control of the stimulation condition (left panel). The unstimulated T cell line is shown in the right panel. Shown are the CD8+ T cells from the live lymphocyte gate.
CYTO_22035_sm_SuppInfoFig6B.tif59KOnline Fig.6 Staining and biological controls. A T cell line from a human melanoma tumor was stimulated with allogeneic tumor. The intracellular cytokines were blocked using purified Ab (IL-4, IL-10, TNF, IFN-γ) prior to staining with fluorochrome-conjugated Ab or the fluorochrome-conjugated Ab was blocked with recombinant human cytokine (IL-2) prior to addition to the cells (lower panels). The cytokine+ gates from the blocked sample are copied onto the unblocked sample for determination of the frequency of cytokine+ cells induced by the stimulation (upper panels). Shown are the CD4+ T cells from the live lymphocyte gate. B T cell line with known IFN-γ response to autologous tumor included as a biological control of the stimulation condition (left panel). The unstimulated T cell line is shown in the right panel. Shown are the CD8+ T cells from the live lymphocyte gate.
CYTO_22035_sm_SuppInfoFig7.tif450KOnline Fig.7 48-hr stability of staining. PBMC from a healthy donor were stimulated with PMA and Ionomycin for 4 hours followed by staining as outlined in the OMIP. Samples were acquired fresh (Day 1), or were stored at 4°C for either 24 hours (Day 2) or 48 hours (Day 3) before acquisition. Upper panels show the stability of the APC-Cy7 CD4+ and QD605 CD8+ T cell staining. Bottom panels show the stability of the different cytokine staining.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.