cd8+ t cell depletion kit  (STEMCELL Technologies Inc)

Journal: PLoS ONE

Article Title: The Use of Wavelength Modulated Raman Spectroscopy in Label-Free Identification of T Lymphocyte Subsets, Natural Killer Cells and Dendritic Cells

doi: 10.1371/journal.pone.0125158

Figure Lengend Snippet: (a) CD4 staining of isolated CD4 + T cells. (b) IL-2 ELISA of CD4 + T cells stimulated with or without anti-CD3/CD28 beads. (c) CD8 staining or isolated CD8 + T cells. (d) IFNγ ELISPOT assay of PBMC and purified CD8 + T cells incubated with and without EBV derived peptide AVFDRKSDAK. (e) CD56 staining of isolated NK cells. (f) NK cell degranulation assay—CD107a staining of NK cells incubated without (left panel) or with (right panel) MHC class I deficient. 221 cells at a 10:1 effector to target ratio. (g) CD303 staining of isolated pDC. (h) CD1c staining of isolated mDC. The x-axis in each flow cytometry plot indicates fluorescent intensity. The left hand peak in each flow cytometry plot indicates control staining with an irrelevant antibody. Representative white-light microscopy images of each of the purified cell populations used in Raman spectroscopy experiments are also shown.

Article Snippet: Cells were isolated using Dynabeads (Life Technologies) untouched human CD4 T cell kit (depleting antibodies comprising anti-CD8, CD14, CD16a, CD16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human CD8 T cell kit (depleting antibodies comprising anti-CD4, CD14, CD16a, Cd16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human NK cell kit (depleting antibodies comprising anti-CD3, CD14, CD36, HLA Class II, CD123 and CD235a).

Techniques: Staining, Isolation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Purification, Incubation, Derivative Assay, Degranulation Assay, Flow Cytometry, Light Microscopy, Raman Spectroscopy

Journal: PLoS ONE

Article Title: The Use of Wavelength Modulated Raman Spectroscopy in Label-Free Identification of T Lymphocyte Subsets, Natural Killer Cells and Dendritic Cells

doi: 10.1371/journal.pone.0125158

Figure Lengend Snippet: (a) Mean standard Raman spectra of CD4 + , CD8 + and CD56 + NK cells. (b)-(d) Pairwise comparison of the WMRS spectra obtained from purified lymphocyte subsets. (b) Mean spectra of CD4 + versus CD8 + T cells. (c) Mean spectra of CD4 + T cells versus CD56 + NK cells. (d) Mean spectra of CD8 + T cells versus CD56 + NK cells. Solid spectra lines represent mean of each subset, with shadow lines representing the standard deviation. Shaded vertical bands indicated regions of significant difference, estimated by student’s T test at level of p<10 –7 .

Article Snippet: Cells were isolated using Dynabeads (Life Technologies) untouched human CD4 T cell kit (depleting antibodies comprising anti-CD8, CD14, CD16a, CD16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human CD8 T cell kit (depleting antibodies comprising anti-CD4, CD14, CD16a, Cd16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human NK cell kit (depleting antibodies comprising anti-CD3, CD14, CD36, HLA Class II, CD123 and CD235a).

Techniques: Purification, Standard Deviation

Journal: PLoS ONE

Article Title: The Use of Wavelength Modulated Raman Spectroscopy in Label-Free Identification of T Lymphocyte Subsets, Natural Killer Cells and Dendritic Cells

doi: 10.1371/journal.pone.0125158

Figure Lengend Snippet: (a) CD4 + , CD8 + T cells and CD56 + NK cells. (b) CD4 + and CD8 + T cells. (c) CD4 + T cells and CD56 + NK cells. (d) CD8 + T cells and CD56 + NK cells. (3D rotating views of these plots are available to view in the supplementary information).

Article Snippet: Cells were isolated using Dynabeads (Life Technologies) untouched human CD4 T cell kit (depleting antibodies comprising anti-CD8, CD14, CD16a, CD16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human CD8 T cell kit (depleting antibodies comprising anti-CD4, CD14, CD16a, Cd16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human NK cell kit (depleting antibodies comprising anti-CD3, CD14, CD36, HLA Class II, CD123 and CD235a).

Techniques:

Journal: PLoS ONE

Article Title: The Use of Wavelength Modulated Raman Spectroscopy in Label-Free Identification of T Lymphocyte Subsets, Natural Killer Cells and Dendritic Cells

doi: 10.1371/journal.pone.0125158

Figure Lengend Snippet: Confusion matrix for CD4 + , CD8 + and CD56 + cell subsets.

Article Snippet: Cells were isolated using Dynabeads (Life Technologies) untouched human CD4 T cell kit (depleting antibodies comprising anti-CD8, CD14, CD16a, CD16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human CD8 T cell kit (depleting antibodies comprising anti-CD4, CD14, CD16a, Cd16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human NK cell kit (depleting antibodies comprising anti-CD3, CD14, CD36, HLA Class II, CD123 and CD235a).

Techniques:

Journal: PLoS ONE

Article Title: The Use of Wavelength Modulated Raman Spectroscopy in Label-Free Identification of T Lymphocyte Subsets, Natural Killer Cells and Dendritic Cells

doi: 10.1371/journal.pone.0125158

Figure Lengend Snippet: (a) CD4 + T cells. (b) CD8 + T cells. (c) CD56 + NK cells. (3D rotating views of these plots are available to view in the supplementary information).

Article Snippet: Cells were isolated using Dynabeads (Life Technologies) untouched human CD4 T cell kit (depleting antibodies comprising anti-CD8, CD14, CD16a, CD16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human CD8 T cell kit (depleting antibodies comprising anti-CD4, CD14, CD16a, Cd16b, CD19, CD36, CD56, CD123 and CD235a), Dynabeads untouched human NK cell kit (depleting antibodies comprising anti-CD3, CD14, CD36, HLA Class II, CD123 and CD235a).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Antigen-specific clonal expansion and cytolytic effector function of CD8 + T lymphocytes depend on the transcription factor Bcl11b

doi: 10.1084/jem.20092136

Figure Lengend Snippet: Removal of Bcl11b in mature T cells with the dLck-iCre deleter. (A) Flow cytometry analysis of Bcl11b in gated CD4 + and CD8 + T lymphocytes from spleen, and in CD4 and CD8 SP and DP thymocytes of Bcl11b F/F /dLck-iCre (KO) and control mice (WT). Single cell suspensions of spleens and thymi were stained for CD4, CD8, and Bcl11b. This is a representative experiment out of three, with three pairs of mice. (B) FACS analysis of thymocytes of Bcl11b F/F /dLck-iCre (KO) and control mice (WT). (C) FACS analysis of T cells from spleens and lymph nodes of Bcl11b F/F /dLck-iCre (KO) and control mice (WT) and spleens from Bcl11b F/F /dLck-iCre/OT-1 and OT-1 control mice. (D) Total cellularity of CD8 + T cells. Absolute numbers were derived based on the percentage of CD8 + T cells in total live cells. A two-tailed Student’s t test was applied to determine statistical significance. P = 0.0133. In B–D, data are derived from 4–10 pairs of mice. Error bars indicate SD.

Article Snippet: Naive CD8 + T lymphocytes were either sorted as CD62L hi CD44 lo population or purified by depletion, using the CD8 + T cell depletion kit (Miltenyi Biotec).

Techniques: Flow Cytometry, Staining, Derivative Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Antigen-specific clonal expansion and cytolytic effector function of CD8 + T lymphocytes depend on the transcription factor Bcl11b

doi: 10.1084/jem.20092136

Figure Lengend Snippet: Bcl11b-deficient CD8 + T cells have a defect in expansion during the immune response to Lm-Ova and influenza infection. (A) Expansion of Bcl11b-deficient and WT OT-1 CD8 + T cells in spleen during the course of infection with LM-Ova. Naive CD62L hi CD44 lo CD8 + T lymphocytes from CD45.2 Bcl11b F/F /dLck-iCre/OT-1 and CD45.1/2/OT-1 mice (1:1; total 10 4 ) were cotransferred in CD45.1 recipient mice. The next day after transfer, mice were infected with 1.5 × 10 4 CFU Lm-Ova, and the immune response was monitored by evaluating the frequency of CD45.2 (Bcl11b deficient) and CD45.1/2 (WT) CD8 + T cells on days 6, 7, 9, and 12 after infection. This is representative data from four independent experiments in which cells were transferred from three mice from each group into three to four recipients. (B) Graph depicting the expansion of Bcl11b-deficient and WT OT-1 CD8 + T cells in spleen at the indicated time points after Lm-Ova infection, as described in A. Data are expressed as mean ± SD of the percentage of donor CD8 + T lymphocytes and a mean of four independent experiments. (C) Influenza-specific Bcl11b-deficient and WT CD8 + T cells in the mediastinal lymph nodes on day 10 after influenza infection. Mixed bone marrow chimeras were generated by reconstitution of lethally irradiated CD45.1 with either a 1:1 mix of CD45.1 and CD45.2 bone marrows (WT/WT chimeras) or a 1:1 mix of CD45.1 and Bcl11b F/F /dLck-iCre bone marrows (WT/KO chimeras). After reconstitution, mice were infected with 1,000 EIU PR8. The frequencies of NP-specific and PA-specific CD8 + T cells derived from each donor type were determined by flow cytometry. The numbers in each plot refer to the mean percentage ± SD of CD8 + T cells that are either NP- or PA-specific. Data are representative for six pairs of donor mice, in which there were five mice reconstituted from each pair.

Article Snippet: Naive CD8 + T lymphocytes were either sorted as CD62L hi CD44 lo population or purified by depletion, using the CD8 + T cell depletion kit (Miltenyi Biotec).

Techniques: Infection, Generated, Irradiation, Derivative Assay, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Antigen-specific clonal expansion and cytolytic effector function of CD8 + T lymphocytes depend on the transcription factor Bcl11b

doi: 10.1084/jem.20092136

Figure Lengend Snippet: Proliferation and survival of Bcl11b-deficient OT-1 CD8 + T cells in vivo after Ag-specific activation. (A) Proliferation at day 6 after infection was evaluated by BrdU incorporation 16 h after the i.p. injection of BrdU in mice cotransferred with KO and WT cells and then infected with Lm-Ova, as described in . BrdU incorporation was evaluated within the donor Bcl11b-deficient CD45.2 and WT CD45.1/2 CD8 + T cell populations. (B) Forward scatter of the CD8 + T cells was evaluated in the same conditions as in A. (C) Survival of Bcl11b-deficient and WT CD8 + T cells during the course of infection with Lm-Ova. Cleaved caspase-3 was evaluated within the donor Bcl11b-deficient CD45.2 and WT CD45.1/2 CD8 + T cell populations at the indicated time points. Bcl11b-deficient CD45.2 and WT CD45.1/2 CD8 + T cells were transferred in CD45.1 recipient mice further infected with Lm-Ova, as described in . Histograms show levels of cleaved caspase-3 on gated CD45.2 (Bcl11b-deficient) or CD45.1/2 (WT) CD8 + T cell populations versus isotype control (filled histogram). Numbers indicate the percentage of cells within the gated populations. Levels of cleaved caspase-3 in recipient CD8 + T cells are indicated in Fig. S3 as an internal control. Data in A–C are representative of three independent experiments.

Article Snippet: Naive CD8 + T lymphocytes were either sorted as CD62L hi CD44 lo population or purified by depletion, using the CD8 + T cell depletion kit (Miltenyi Biotec).

Techniques: In Vivo, Activation Assay, Infection, BrdU Incorporation Assay, Injection

Journal: The Journal of Experimental Medicine

Article Title: Antigen-specific clonal expansion and cytolytic effector function of CD8 + T lymphocytes depend on the transcription factor Bcl11b

doi: 10.1084/jem.20092136

Figure Lengend Snippet: In vitro proliferation of Bcl11b-deficient and WT CD8 + T cells in response to TCR stimulation. (A) Bcl11b-deficient and WT naive OT-1 CD8 + T cells were labeled with CFSE and incubated for 3 d with 0.1 µM OVA 257-264 peptide-loaded APCs. Numbers of cell divisions of WT or knockout cells were measured by CFSE dilution. The frequency of cells in each CFSE dilution peak is given. Data are representative of three independent experiments. (B) Response of Bcl11b-deficient and WT naive OT-1 CD8 + T lymphocytes to TCR stimulation. Bcl11b-deficient and WT naive OT-1 CD8 + T cells were cultured with OVA 257-264 peptide-pulsed APCs for 5 h. Cells were then stained for CD8 and CD69 and analyzed by flow cytometry. Histograms represent surface levels of CD69 within the gated CD8 + T cells. Data are representative of three independent experiments, in which two to three pairs of mice were used. (C and D) In vitro proliferation in response to stimulation with α-CD3/CD28 antibodies (C) and PMA/ionomycin (D), evaluated as in A. Data are representative of two independent experiments with two pairs of mice.

Article Snippet: Naive CD8 + T lymphocytes were either sorted as CD62L hi CD44 lo population or purified by depletion, using the CD8 + T cell depletion kit (Miltenyi Biotec).

Techniques: In Vitro, Labeling, Incubation, Knock-Out, Cell Culture, Staining, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Antigen-specific clonal expansion and cytolytic effector function of CD8 + T lymphocytes depend on the transcription factor Bcl11b

doi: 10.1084/jem.20092136

Figure Lengend Snippet: Bcl11b regulates expression of CD8 coreceptor and Plcγ1 genes. (A) Surface and intracellular levels of CD8α and CD8β coreceptors. Single cell suspensions from lymph nodes of Bcl11b F/F /dLck-iCre and control mice were stained for surface or intracellular CD8α and CD8β. Overlayed histograms indicate surface or intracellular CD8α and CD8β. CD8α and CD8β MFIs are also indicated. Representative data for five pairs of mice are shown. (B) Relative mRNA levels of the CD8 coreceptor in sorted naive CD44 lo Bcl11b-deficient and WT CD8 + T lymphocytes from four (CD8a) and three (CD8b) pairs of mice, normalized to actin. A two-tailed Student’s t test was applied to determine statistical significance. P = 3.66 × E −4 , and 0.006. (C) Immunoblot analysis of Zap70 phosphorylation after TCR, CD8, and CD28 cross-linking of purified CD8 + T cells from OT1 mice. CD8 + T cells were purified by depletion, and CD8 + T cells were preincubated with the indicated biotinylated antibodies, followed by addition of streptavidin. 10 6 cells were used in each treatment. P-Zap70 was quantified by densitometric analysis versus total Zap70. Data are representative for two pairs of mice. (D) Relative Plcg1 mRNA levels in Bcl11b-deficient (KO) and WT (WT) CD8 + T cells from four pairs of mice, normalized to actin. * indicates significant difference. Error bars indicate SD. P = 0.05. (E) Calcium flux analysis in Bcl11b-deficient (KO) and WT (WT) CD8 + T cells. Lymphocytes were loaded with Fluo4 and surface stained for CD4 and CD8. Cells were preincubated with biotinylated α-CD3/CD28, after which streptavidin was added (arrow), and the fluorescence was measured for the indicated period of time. The histogram on the right indicates the fluorescence after addition of ionomycin to record the maximal calcium response. Calcium flux was analyzed using the kinetics platform of FlowJo and represented as Fluo4 fluorescence over time. This is a representative experiment out of three.

Article Snippet: Naive CD8 + T lymphocytes were either sorted as CD62L hi CD44 lo population or purified by depletion, using the CD8 + T cell depletion kit (Miltenyi Biotec).

Techniques: Expressing, Staining, Two Tailed Test, Western Blot, Purification, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Antigen-specific clonal expansion and cytolytic effector function of CD8 + T lymphocytes depend on the transcription factor Bcl11b

doi: 10.1084/jem.20092136

Figure Lengend Snippet: BCL11B associates with the E8I, E8IV, and E8V enhancers. ChIPs with anti-Bcl11b or IgG antibodies on purified CD8 + T cells. The association of Bcl11b with the CD8 enhancers was evaluated by qPCR using previously described primers , which cover the indicated regions of the enhancers. Binding is expressed relative to the input and calculated as indicated in Materials and methods. Data are representative of four independent experiments with four independent pairs of mice.

Article Snippet: Naive CD8 + T lymphocytes were either sorted as CD62L hi CD44 lo population or purified by depletion, using the CD8 + T cell depletion kit (Miltenyi Biotec).

Techniques: Purification, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Antigen-specific clonal expansion and cytolytic effector function of CD8 + T lymphocytes depend on the transcription factor Bcl11b

doi: 10.1084/jem.20092136

Figure Lengend Snippet: Evaluation of bacterial burden and Ag-specific cytolytic activity in the absence of Bcl11b from CD8 + T cells. (A) Bacterial burden was assessed in the livers of Lm-Ova–infected mice, separately transferred with Bcl11b-deficient and WT OT-1 CD8 + T cells, on days 3, 4, and 5 after infection. CFUs were normalized to milligrams of tissue. Three independent experiments were conducted, each with three pairs of mice. (B) Ex vivo killing of Ag-specific targets by Bcl11b-deficient and WT OT-1 CD8 + T cells was evaluated by measuring lactate dehydrogenase activity released into the media after killing of target cells, as described in the Materials and methods. Bcl11b-deficient and WT OT-1 CD8 + T cells were purified from spleens of Lm-Ova–infected mice on day 5 after infection and incubated with MEC.B7.SigOva cells expressing OVA 257-264 peptide via MHC class I . Data are presented as means ± SD of cytotoxicity (lactate dehydrogenase activity) of triplicate samples and are representative of three independent experiments.

Article Snippet: Naive CD8 + T lymphocytes were either sorted as CD62L hi CD44 lo population or purified by depletion, using the CD8 + T cell depletion kit (Miltenyi Biotec).

Techniques: Activity Assay, Infection, Ex Vivo, Purification, Incubation, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Antigen-specific clonal expansion and cytolytic effector function of CD8 + T lymphocytes depend on the transcription factor Bcl11b

doi: 10.1084/jem.20092136

Figure Lengend Snippet: Evaluation of effector population and effector molecules in the absence of Bcl11b from CD8 + T cells. (A) The formation of effector population during the immune response to Lm-OVA infection was evaluated in Bcl11b-deficient and WT donor OT-1 CD8 + T cells on days 6, 7, 9, and 12 after infection by staining for Klrg1 and CD127. The boxes indicate the Klrg1 hi CD127 lo and Klrg1 lo CD127 hi populations gated on the donor CD45.2 Bcl11b-deficient and CD45.1/2/ WT CD8 + T cells. Data are representative of three independent experiments. (B) Granzyme B was evaluated on day 7 after the Lm-Ova infection within the donor Bcl11b-deficient CD45.2 and WT CD45.1/2 CD8 + T cell populations. Experiments were conducted as described in . Numbers indicate the percentage of cells in the gated populations. The histograms on the right show granzyme B in CD8 + T cells from uninfected mice. Data are representative of three independent experiments in which cells were transferred from two to three mice from each group in three to four recipients. (C) Western blot analysis of perforin in purified CD8 + T cells from mice infected with Lm-Ova. Bcl11b F/F /dLck-iCre/OT1 and WT OT1 mice were infected with Lm-Ova, and 7 d after infection total CD8 + T cells were purified. Extracts were prepared from equal numbers of cells and Western blot analysis was conducted for evaluation of perforin. Data are representative of two independent experiments. (D) Relative mRNA levels of perforin and granzyme B (normalized to actin) in equal numbers of total CD8 + T cells purified form LM-Ova–infected Bcl11b F/F /dLck-iCre/OT1 and WT OT1 mice. Data are derived from multiple experiments with, respectively, 10 and 11 pairs of mice and, respectively, P = 0.0039 and 0.0001. * indicates significant difference. (E and F) ChIP assays with anti-Bcl11b or anti-GFP antibodies on purified CD8 + T cells from infected mice at day 6 after infection. Association of Bcl11b with perforin (E) and granzyme B (F) genomic regions was evaluated by qPCR using primers covering the indicated regions upstream of TSS, and values were expressed relative to the input as described in Materials and methods. Data are representative of two independent experiments on two pairs of mice. (G and H) Evaluation of perforin and granzyme B mRNA levels in CD8 + T cells from Bcl11b F/F /ER-Cre and ER-Cre mice in vitro stimulated for 2 d, before the initiation of the treatment with OH-tamoxifen, according to a protocol for in vitro differentiation to effector cells . After activation, cells were grown in the presence of 100 U/ml rIL-2 and 100 nM OH-tamoxifen for 4–5 d and RNA was purified followed by qRT-PCR. This is representative of two independent experiments, each with two to three pairs of mice. P = 0.64 and 0.46, respectively. Error bars indicate SD.

Article Snippet: Naive CD8 + T lymphocytes were either sorted as CD62L hi CD44 lo population or purified by depletion, using the CD8 + T cell depletion kit (Miltenyi Biotec).

Techniques: Infection, Staining, Western Blot, Purification, Derivative Assay, In Vitro, Activation Assay, Quantitative RT-PCR