Production of cell culture‐derived infectious HCV (HCVcc) tagged with Gaussia luciferase from the Jc1FLAG2(p7-nsGluc2A) construct (genotype 2a, provided by Dr. Charles M. Rice) has been described elsewhere27 (link). HCV viral titer was expressed as focus forming units (FFU) and determined by immunofluorescence staining using primary anti-HCV NS5A mouse antibody (1:25,000; clone 9E10, gift from Dr. Charles M. Rice), followed by Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG) (H+L) (1:400; Invitrogen) as previously reported27 (link).
Alexa fluor 488 goat anti mouse immunoglobulin g igg h l
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG) (H+L) is a secondary antibody used for detection and visualization in immunoassays and immunofluorescence applications. It is a polyclonal antibody raised in goat that specifically binds to the heavy and light chains of mouse IgG antibodies. The antibody is conjugated to the Alexa Fluor 488 fluorescent dye, which emits green fluorescence upon excitation.
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2 protocols using alexa fluor 488 goat anti mouse immunoglobulin g igg h l
1
Production and Quantification of HCVcc
2
Multicolor Flow Cytometry for Porcine T-Cell Analysis
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Flow cytometry was conducted to analyze the frequency of CD4+ cells, CD8+ cells,
interferon γ (IFN-γ), and the ratio of CD4+/CD8+. Cells were stained with 10 µL
of anti-porcine CD25 (AbD Serotec, MCA1736 clone K231.3B2), followed by 0.06 µg
of Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG, H+L; Invitrogen,
Molecular Probes, Carlsbad, CA, USA). The LYNX rapid antibody conjugation kit
(AbD Serotec) was used to conjugate mouse anti-porcine CD4 with allophycocyanin
(APC; clone 74-12-4; VMRD, Inc., Pullman, Wa, USA) and CD8 with RPECy7 (clone
76-2-11; VMRD, Inc.), and 0.05 µg of these antibodies were then added to the
cell preparation. Forkhead box P3 (Foxp3) intracellular staining was performed
with 0.03 µg of anti-rat/mouse Foxp3 (clone FJK-16s, with cross-reactivity with
swine Foxp3; eBioscience, San Diego, CA, USA) and 0.03 µg of PE-conjugated rat
IgG2a isotype control (clone eBR2a; eBioscience) using the Foxp3 Staining Buffer
Set (Staining, Fixation/Permeabilization, and Permeabilization Buffers;
eBioscience) to obtain the four-color stain
CD4APCCD8RPECy7CD25Alexa488Foxp3PE.
The frequency of regulatory T cells (Tregs) was evaluated by flow cytometry (BD
FACS Canto II), and data were analyzed using BD FACS Diva 6.0 software.
interferon γ (IFN-γ), and the ratio of CD4+/CD8+. Cells were stained with 10 µL
of anti-porcine CD25 (AbD Serotec, MCA1736 clone K231.3B2), followed by 0.06 µg
of Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG, H+L; Invitrogen,
Molecular Probes, Carlsbad, CA, USA). The LYNX rapid antibody conjugation kit
(AbD Serotec) was used to conjugate mouse anti-porcine CD4 with allophycocyanin
(APC; clone 74-12-4; VMRD, Inc., Pullman, Wa, USA) and CD8 with RPECy7 (clone
76-2-11; VMRD, Inc.), and 0.05 µg of these antibodies were then added to the
cell preparation. Forkhead box P3 (Foxp3) intracellular staining was performed
with 0.03 µg of anti-rat/mouse Foxp3 (clone FJK-16s, with cross-reactivity with
swine Foxp3; eBioscience, San Diego, CA, USA) and 0.03 µg of PE-conjugated rat
IgG2a isotype control (clone eBR2a; eBioscience) using the Foxp3 Staining Buffer
Set (Staining, Fixation/Permeabilization, and Permeabilization Buffers;
eBioscience) to obtain the four-color stain
CD4APCCD8RPECy7CD25Alexa488Foxp3PE.
The frequency of regulatory T cells (Tregs) was evaluated by flow cytometry (BD
FACS Canto II), and data were analyzed using BD FACS Diva 6.0 software.
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