Lungs were minced into small pieces and allowed to digest with gentle agitation (80 rpm, 37°C, 45 min) onto canonical tubes containing 5 ml of digestion buffer (0.5 mg/ml collagenase and 20 μg/ml DNase diluted in RPMI medium). After, the cell suspension was passed through the cell strainer (pore size, 70 μm; BD Biosciences, San Jose, CA), and the remaining erythrocytes were lysed with ACK buffer (Invitrogen, Carlsbad, CA). Cells (1 × 106) were blocked with mouse BD FC Block (5 μg/ml, catalog no. 553141; BD Pharmingen) and then stained using fluorescent-labeled monoclonal antibodies, as follows: Ly6G-BV421 (1:200, catalog no. 127627; BioLegend); CD45-fluorescein isothiocyanate (FITC) (1:200, catalog no. 5530801; BD Pharmingen); F4/80-phycoerythrin (PE)-Cyanine 7 (1:100, catalog no. 25-4801-82; Invitrogen); CD4-PE (1:100, catalog no. 100408; BioLegend); CD3-PerCP-Cyanine 5.5 (1:100, catalog no. 100218; BioLegend); CD8-allophycocyanin (APC) (1:100, catalog no. 17-0081-82; Invitrogen). The acquisition was carried out in a BD FACSCanto II cell analyzer and analyzed using FlowJo software (Tree Star, Ashland, OR).
Cd8 allophycocyanin apc
Manufactured by Thermo Fisher Scientific
CD8-allophycocyanin (APC) is a fluorescent dye-conjugated antibody used in flow cytometry applications. It binds to the CD8 cell surface antigen, allowing for the identification and enumeration of CD8-positive cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 pe, by BioLegend (1 mentions)
Ly6g bv421, by BioLegend (1 mentions)
Mouse fc block, by BD (1 mentions)
Ack buffer, by Thermo Fisher Scientific (1 mentions)
Cd45 fluorescein isothiocyanate fitc, by BD (1 mentions)
Cd3 percp cyanine5, by BioLegend (1 mentions)
Cell strainer, by BD (1 mentions)
Isotype control, by BD (1 mentions)
Fix and perm, by Thermo Fisher Scientific (1 mentions)
Fc500 mpl, by Beckman Coulter (1 mentions)
Cd3 ecd, by Beckman Coulter (1 mentions)
2 protocols using cd8 allophycocyanin apc
1
Lung Cell Isolation and Immunophenotyping
2
Profiling Cytotoxic T Cell Responses
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MNC cultured for 3 days in the presence of flu antigens were fixed and permeabilized (Fix and Perm; Invitrogen) according to the manufacturer's instructions before staining with either a ‘positive’ or a ‘negative’ antibody cocktail. The ‘positive’ mixture comprised CD3-ECD (Beckman Coulter), CD8-allophycocyanin (APC) (Invitrogen), anti-perforin-PE and anti-granzyme-FITC (both from Becton–Dickinson). The ‘negative’ mixture contained the same antibodies except for isotype controls (Becton Dickinson) as substitutes for perforin and granzyme antibodies. Cells were analyzed by flow cytometry (Beckman Coulter FC500 MPL).
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