Spleen and lymph node cells were purified by forcing tissue through 40 μm mesh into complete RPMI media (Gibco) containing 6% serum. PE-specific B cells were enriched as previously described (Pape et al., 2011 (link); Taylor et al., 2012b (link)). To enrich plasma cells, cell suspensions were made from bone marrow in PBS containing 1 mM EDTA and 2% serum. Anti-CD138-APC (BD Biosciences) and anti-APC microbeads (Militenyi Biotec) were used for enrichment with magnetized LS columns. In figure 3 , APC-specific cells were enriched by negative selection using anti-CD4-biotin, anti-CD8-biotin, anti-Gr1-biotin, and anti-Ter119-biotin (eBioscience), streptavidin microbeads (Militenyi Biotec), and magnetized LS columns. Following column-based enrichment, samples were processed for flow cytometry. Cell counts from column-bound and flow-through samples were assessed using AccuCheck counting beads (Invitrogen) and cell numbers from both fractions were evaluated.
Anti cd4 biotin
Manufactured by Thermo Fisher Scientific
Anti-CD4-biotin is a biotinylated antibody that binds to the CD4 cell surface receptor. It can be used for the detection and isolation of CD4-positive cells in various research and diagnostic applications.
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Lab products found in correlation
Anti ter119 biotin, by Thermo Fisher Scientific (1 mentions)
Complete rpmi media, by Thermo Fisher Scientific (1 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Anti cd138 apc, by BD (1 mentions)
Lsrii fortessa flow cytometer, by BD (1 mentions)
Anti cd8 fitc, by Thermo Fisher Scientific (1 mentions)
Pe anti mouse ifn γ, by BioLegend (1 mentions)
Streptavidin pe cy5, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
2 protocols using anti cd4 biotin
1
Purification and Enrichment of Immune Cells
2
Quantitative Analysis of Virus-Specific T-Cells
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The lung and BALF cell samples were harvested at 6 days post infection from the mice immunized and challenged with A/Nanchang/H3N2 virus. The isolated lung and BALF cells were in vitro culture-stimulated with 5 µg/ml of M2e peptide and/or stalk protein with Golgi stopper (2 µg/mL). After 5 h culture, the cells were stained with mouse anti-CD3-PacificBlue (300x dilution, cat no: 100214, clone 17A2, BioLegend), anti-CD4-biotin (300x dilution, cat no: 13-0042-85, clone RM4-5, eBioscience), streptavidin-PE/Cy5 (300x dilution, cat no: 15-4317-82, eBioscience), and anti-CD8-FITC (300x dilution, cat no: MA5-16759, clone 53–6.7, eBiosciences), followed by fixation and permeabilization using a Cytofix/Cytoperm kit (10x dilution, cat no: 554715, BD Biosciences) and then, staining of intracellular cytokine using IFN-γ mAb (anti-mouse IFN-γ-PE, 200x dilution, cat no: 505808, clone XMG1.2, BioLegend). The IFN-γ+ T-cells were acquired by Becton-Dickinson LSR-II/Fortessa flow cytometer (BD) and analyzed by a sequential lymphocyte gating strategy (Supplementary Fig. S10 ) using FlowJo software (FlowJo V10, Tree Star, Inc.)45 .
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