CD4+ T cells from the spleen of mice were purified using LS positive selection MACs and anti‐CD4 (L3T4) beads (Miltenyi Biotec). CD4+ MACs sorted cells were further purified by FACS using a FACS Aria (BD Biosciences) with a 70 μm nozzle insert and FACS Diva software. CD4+ MACs sorted cells were stained for 20 min at 4°C in the dark with the following antibodies: anti‐CD4‐PerCPCy5.5 (RM4‐5), anti‐CD25‐PE (PC61), anti‐CD62L‐PECy7 (MEL‐14; Thermo Fisher), and anti‐CD44‐Pacific Blue (IM1.8.1; Thermo Fisher). Single‐positive compensation controls and unstained controls were used to set up instrument settings and for gating strategies. Cell purity was verified post‐sort (requiring 95% purification) and cell viability was assessed using trypan‐blue staining. Sorted cells were collected in complete media (as described above), counted using a hemocytometer, and immediately cultured or transferred in vivo.
Anti cd4 l3t4 beads
Manufactured by Miltenyi Biotec
Anti-CD4 (L3T4) beads are a laboratory product designed for the isolation and separation of CD4-positive cells. They consist of magnetic beads coated with antibodies that specifically bind to the CD4 cell surface antigen. These beads can be used to isolate CD4+ cells from various biological samples for research or analytical purposes.
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2 protocols using anti cd4 l3t4 beads
1
Purification of Murine CD4+ T Cells
2
Purification and Characterization of Murine CD4+ T Cells
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CD4 + cells from the spleen of mice were purified using LS positive selection magneticactivated columns (MACs) and anti-CD4 (L3T4) beads (Miltenyi Biotec). CD4 + MACs sorted cells were further purified by fluorescence activated cell sorting using a FACS Aria (BD Biosciences) with a 70µm nozzle insert and FACS Diva software CD4 + MACs sorted cells were stained for 20 mins at 4°C in the dark with the following antibodies: anti-CD4-PerCPCy5.5 (RM4-5), anti-CD25-PE (PC61), anti-CD62L-PECy7 (MEL-14; Thermo Fisher) and anti-CD44-Pacific Blue (IM1.8.1; Thermo Fisher). Single positive compensation controls and unstained controls were used to set up instrument settings and for gating strategies. Cell purity was verified post-sort (requiring 95% purify) and cell viability assessed using trypanblue staining. Sorted cells were collected in complete media (as described above) and after cells were counted using a haemocytometer they were immediately cultured or transferred in vivo.
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