LP cells from colon or splenic immune cells were sorted using a FACS flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo_V10 (Treestar, Ashlan, OR). The gating strategy has been shown in supplementary Figure 8. For macrophage and neutrophil analyses, cells were incubated with the following antibodies: FITC-conjugated anti-CD11b (11-0112-82, eBioscience, San Diego, CA), APC-conjugated anti-CD11c (17-0114-81, eBioscience), PE-cyanine7-conjugated anti-F4/80 (25-4801-82, eBioscience), PE-conjugated anti-CD206 (12-2061-80, eBioscience), and PE-conjugated anti-Ly6G (12-5931-81, eBioscience); for T cell analyses, cells were incubated with the following antibodies: APC-conjugated anti-CD3 (17-0032-82, eBioscience), FITC-conjugated anti-CD4 (11-0042-81, eBioscience), and PE-conjugated anti-CD8 (12-0081-81, eBioscience); for B cell and NK cell analyses, cells were incubated with the following antibodies: FITC-conjugated anti-CD19 (11-0193-81, eBioscience), and PE-conjugated anti-NK1.1 (12-5941-81, eBioscience).
Pe conjugated anti cd8
Manufactured by Thermo Fisher Scientific
Sourced in United States
PE-conjugated anti-CD8 is a fluorochrome-labeled antibody that recognizes the CD8 antigen, a cell surface glycoprotein expressed on a subset of T cells. This product is designed for use in flow cytometry applications to identify and analyze CD8-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated anti cd4, by Thermo Fisher Scientific (2 mentions)
Fitc conjugated anti cd3, by Thermo Fisher Scientific (2 mentions)
Pe conjugated anti nk1, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd11c, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd11b, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd3, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd19, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti ly6g, by Thermo Fisher Scientific (1 mentions)
Facs flow cytometer, by BD (1 mentions)
Pe cy5 conjugated anti cd3, by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Dnase, by Merck Group (1 mentions)
Apc conjugated anti cd4, by Thermo Fisher Scientific (1 mentions)
Counting beads, by Spherotech (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Pe conjugated anti cd4, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti igm, by BD (1 mentions)
Fitc conjugated anti cd45r, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by BD (1 mentions)
5 protocols using pe conjugated anti cd8
1
Flow Cytometry Analysis of Immune Cell Subsets
2
Flow Cytometry Analysis of Lacrimal Gland Cells
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The flow cytometry strategy for LG cultures has been described previously.25 (link),26 (link) Briefly, single-cell suspensions of four LGs from each condition were prepared by treating minced tissue fragments with 100 U/mL collagenase D and 15 µg/mL DNase (Sigma-Aldrich) for 40 minutes at 37°C. After blocking with 1 µg unlabeled anti-FcrR antibody (clone 2.4G2), cells (1 × 106) were washed with RPMI 1640 and surface-stained with PE-Cy5–conjugated anti-CD3, FITC-conjugated anti-CD4, and PE-conjugated anti-CD8 (eBioscience, San Diego, CA, USA), and the cells were analyzed using flow cytometry and fluorescence-activated cell sorting (FACS) (FACS Calibur; Becton-Dickinson, Mountain View, CA, USA).
3
Quantification of T Cell Subsets
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After treatment for 24 h at 37°C, FITC-conjugated anti-CD3 (eBioscience, Inc, San Diego, CA, USA), APC-conjugated anti-CD4 (eBioscience), and PE-conjugated anti-CD8 (eBioscience) were incubated with the cells of each group for 1 h at 4°C. Afterwards, the cells were fixed with 1% paraformaldehyde and washed. The percentages of CD3+CD4+ and CD3+CD8+ were calculated using a flow cytometer (BD Biosciences, Franklin lakes, NJ, USA).
4
Immunotherapy for Lung Metastasis
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Mice (n = 6/group) were immunized bi-weekly by intraperitoneal injection with 1 × 106 B6 DC, 1 × 106 DBA DC or PBS. Seven days after the second immunization, 5 × 105 B16 cells were injected into the tail vein. On Day 14, mice were sacrificed and lungs were perfused using PBS. A piece of lung (9.8 ± 1.7 mg) was removed and then digested in 1 mg/mL collagenase IV (Sigma) at 37 °C for 1 h. Dissociated cells were collected through a 70-μm filter and stained with APC-Cy7 conjugated anti-CD45.2, FITC conjugated anti-TCRβ, PerCP conjugated anti-NK1.1, PE conjugated anti-CD8 and APC conjugated anti-KLRG1 antibodies (eBioscience). Flow cytometry (BD FASAria II) was used to collect data and the absolute number of KLRG1+CD8 T cells for each sample was calculated by adding counting beads (Spherotech) to each sample. CD45.2+ cells were gated and analyzed to characterize the tumor-infiltrated lymphocytes.
5
Immunophenotyping of Lymphocyte Subsets
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Cells were doubly incubated with FITC-conjugated anti-CD45R (1 mg/10 6 cells; eBioscience, Thermo Fisher Scientific, Waltham, MA) and PE-conjugated anti-IgM (1 mg/10 6 cells; BD Biosciences, Heidelberg, Germany), FITC-conjugated anti-CD45R (1 mg/10 6 cells; eBioscience) and PE-conjugated anti-CD138 (1 mg/10 6 cells; Abcam, Cambridge, UK), FITC-conjugated anti-CD3 (1 mg/10 6 cells; eBioscience) and PE-conjugated anti-CD4 (0.5 mg/10 6 cells; eBioscience) as well as FITC-conjugated anti-CD3 (1 mg/10 6 cells; eBioscience) and PE-conjugated anti-CD8 (0.125 mg/10 6 cells; eBioscience), respectively. The percentages of
T cells were determined through a lymphocyte gate by flow cytometry (BD Accuri C6, BD Biosciences).
T cells were determined through a lymphocyte gate by flow cytometry (BD Accuri C6, BD Biosciences).
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