The middle and left lobes of the lungs were perfused with 10ml sterile PBS, and digested with 2mg/ml collagenase D at 37°C for 30minutes. Reaction was stopped with the addition of 20µl of 5mM EDTA. Digested tissue was mashed through a 40µm nylon membrane filter to obtain single cell suspensions. Red blood cells were lysed using ACK lysis buffer. Single cell suspensions were surface stained with directly conjugated fluorochrome labeled anti-mouse CD4-V450 (clone RM4-5; BD Horizon), anti-mouse CD8-AF488 (clone 53-6.7; BD Pharmingen), anti-mouse CD44-AF700 (clone IM7; BD Pharmingen) and anti-mouse CD62L-PerCPCy5.5 (clone MEL-14; BD Pharmingen) antibodies. For tetramer staining, lung cells were first surface stained with ESAT6(1–20)-PE tetramer (NIH Tetramer Facility) for 1hr at 37°C, followed by surface staining for T cell markers. Following staining, cells were washed with FACS buffer, fixed in 4% paraformaldehyde and acquired on LSR-II (BD Biosciences). Frequency of specific cell types was calculated using Flow Jo software.
Anti mouse cd4 v450 clone rm4 5
Manufactured by BD
Anti-mouse CD4-V450 (clone RM4-5) is a fluorescent-labeled antibody reagent. It is designed to detect the CD4 cell surface marker on mouse cells.
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Lab products found in correlation
2 protocols using anti mouse cd4 v450 clone rm4 5
1
Isolation and Characterization of Lung T Cells
2
Intracellular Cytokine Profiling of T Cells
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Lung and lymph nodes were examined for intracellular cytokine production. Single cells were obtained as described above. Samples were then stimulated with 10 μg/ml H37Rv cell lysate (BEI NR-14822), 5 μg/ml ESAT6 peptide array (BEI NR-34824), and 5 μg/ml CFP10 peptide array (BEI NR-34825) in RPMI media for 3 h in a 37 °C, 5% CO2 humidified incubator. Stimulated cells were then washed, and surface stained with anti-mouse CD3-FITC (clone 17A2, BD Pharmingen), anti-mouse CD4-V450 (clone RM4-5, BD Horizon), and anti-mouse CD8-PE (clone RM4-5, BD Pharmingen). Subsequently, cells were fixed and permeabilized using BD Cytofix/Cytoperm solution kit (BD 554714) as per the manufacturer’s instructions. Permeabilized cells were stained for intracellular cytokines using anti-mouse IL-17A-AF647 (clone TC11-18H10, BD Pharmingen) and IFNγ-PECy7 (clone XMG1.2, BD Pharmingen). All antibodies were used at a 1:50 dilution. Samples were acquired using the LSRFortessa X-20 and analyzed using FlowJo software (v10.6.1).
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