Journal: PLoS Pathogens

Article Title: Temporal Expression of Bacterial Proteins Instructs Host CD4 T Cell Expansion and Th17 Development

doi: 10.1371/journal.ppat.1002499

Figure Lengend Snippet: C57BL/6 mice were immunized sub-cutaneously with either 100 µg of flagellin 427–441 or SseJ 329–341 peptide in the presence of CFA or infected with 5x10 5 Salmonella (BRD509). On day 7, inguinal, axillary, and brachial lymph nodes were isolated from peptide-immunized mice (Immunized). On day 35, spleens were harvested from C57BL/6 mice infected intravenously with Salmonella BRD509 (Infected). Endogenous flagellin 427–441 - or SseJ 329–341 -specific CD4 T cells were detected using Flagellin:I-A b or SseJ:I-A b tetramers, enriched with anti-fluorochrome microbeads, stained with antibodies to several surface markers, and examined by flow cytometry. CD4 T cells from naive, immunized, and Salmonella -infected mice, were detected among CD11c − CD11b − F4/80 − B220 − CD3 + cells, and further analyzed for expression of CD44 and Flagellin:I-A b or SseJ:I-A b tetramer positive cells. CD4 T cells from the unbound column fraction and CD8 T cells within the bound fraction are also shown as controls. FACS plots are representative of three mice per group and three replicate experiments.

Article Snippet: Cells were incubated with anti-fluorochrome microbeads (Stem cell technologies, Vancouver, Canada) and tetramer-specific cells enriched using a magnet.

Techniques: Infection, Isolation, Staining, Flow Cytometry, Expressing

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: CD4 protein on vaginal lymphoid cells by flow cytometry. Lymph node cell (106) and collagenase-digested vaginal lymphoid-like cells (105) from CBA/J mice were labelled with biotinylated (plus cychrome [CYC]-conjugated streptavidin) RM-4.5 or H129.19 and phycoerythrin (PE)-conjugated RM-4.4 epitope-distinct anti-CD4 antibodies. RM-4.5 and H129.19 anti-CD4 antibodies recognize an epitope in domain 1 of the CD4 protein, whereas RM-4.4 antibodies recognize an epitope in domain 3. Flow cytometric dual staining of lymph node cells (a, c and e) or vaginal lymphoid cells (b, d and f) with RM-4.4 and RM-4.5 anti-CD4 antibodies (c and d) and RM-4.4 and H129.19 anti-CD4 antibodies (e and f) is shown together with isotype control antibodies (a,b). The percentage in each quadrant represents the fluorescent positive cells within the lymphoid-like cell limits. Data shown are a representative of two experiments using 10 mice per experiment.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Flow Cytometry, Staining

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: Immunohistochemical analysis of CD4+ lymph node and vaginal lymphoid cells under non-denaturing conditions. Lymphoid cells isolated from whole lymph nodes or collagenase-treated vaginal tissue from CBA/J mice were incubated with purified RM-4.5 anti-CD4 antibodies or with anti-rat IgG isotype-matched antibodies at 10 µg/ml prior to cytocentrifugation onto slides, and fixation. (a) and (b) show staining of lymph node cells with anti-rat IgG isotype matched antibodies, and RM-4.5 anti-CD4 antibodies, respectively. (c) and (d) show staining of vaginal lymphoid cells with anti-rat IgG isotype-matched antibodies, and RM-4.5 anti-CD4 antibodies, respectively. Arrows on each frame show representative stained cells. The figure is representative of three experiments using different mice.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Immunohistochemical staining, Isolation, Incubation, Purification, Staining

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: Immunohistochemical analysis of CD4+ lymph node and vaginal lymphoid cells under denaturing conditions. Lymph nodes cells or cells collected from collagenase-treated vaginal tissue from CBA/J mice were cytocentrifuged onto slides, fixed and stained with RM-4.5 anti-CD4 antibodies or anti-rat IgG isotype-matched antibodies. (a) and (b) show staining of lymph node cells with anti-rat IgG isotype-matched antibodies, and RM-4.5 anti-CD4 antibodies, respectively. (c) and (d) show staining of vaginal lymphoid cells with anti-rat IgG isotype-matched antibodies, and RM-4.5 anti-CD4 antibodies, respectively. Arrows show representative areas of stained cells. The figure is representative of three experiments using different mice.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: RT-PCR analysis of vaginal T-cell surface marker expression. (a) RT-PCR of total RNA extracted from vaginal lymphoid cells (VL) (5 × 106), whole vaginal tissue (VAG), and lymph node tissue (LN) of CBA/J mice was performed using Taq DNA polymerase and primers designed to amplify CD3 (549 bp), CD8 (513 bp), CD4 (615 bp), CD4B 983 bp, TCR-β (268 bp) and TCR-δ (222 bp) chain constant regions of murine systemic T lymphocytes. Amplification with primers for GAPDH (239 bp) was used as an internal control. The figure is representative of at least five experiments using different mice. (b) Southern blot analysis of CD4 and CD4B amplification products derived from vaginal and lymph node tissue cDNA using a γ-32P-end-labelled probe internal of the predicted amplification product.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Expressing, Amplification, Southern Blot, Derivative Assay

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: lack of CD4 mRNA expression in RM-4.5– RM-4.4+ CD4+ vaginal cells. (a) Vaginal lymphoid cells extracted from CBA/J mice by collagenase-digestion and further purified by Ficoll–Paque® density gradient centrifugation were dual labelled with RM-4.4 and RM-4.5 anti-CD4 antibodies. Those cells that stained positively with RM-4.4 but not RM-4.5 monoclonal antibodies (RM-4.5– RM-4.4+) were FACS sorted. The RM-4.5– RM-4.4+ CD4+ cell-sorted population is represented in the encircled gate of quadrant B4. HeLa cells (5 × 105) were added to the sorted cell population and pelleted. Total RNA was extracted from RM-4.5– RM-4.4+-plus HeLa cells or HeLa cells alone, reverse transcribed and subjected to PCR using a high-efficiency Taq DNA polymerase and primers designed to amplify CD3, CD4, CD4B, TCR β-, and TCR δ chain constant regions of murine systemic T lymphocytes (b). The CD4 and CD4B amplification products shown are from column-purified primary amplification products subjected to a second round of amplification. Amplification with primers for GAPDH was used as an internal control. The figure is representative of two experiments using 40 mice in each experiment.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Expressing, Purification, Gradient Centrifugation, Staining, Amplification