For FACS, dissociated cells were resuspended in cold IsoFlow Sheath Fluid and passed through a 70-μm mesh. Sorting was performed in liquid droplets, using a Cytomation MoFlo Cell Sorting setup at a sheet fluid pressure of 25–30 psi. MMBS for Thy1.2+ cells was performed according to the manufacturer protocol using the Dynabeads Mouse Pan T (Thermo Fisher) kit, with isolation buffer including both BSA and FBS as stated within the manual. To improve cell purity, bead-bound cells were washed twice prior to bead release. In contrast to FACS, which specifically isolates Thy1-GFP+ RGCs, magnetic microbeads directly target the Thy1.2 (CD90.2) surface antigen, crosslinking Thy1-expressing cells to magnetic microbeads, allowing for the enrichment of both GFP+ Thy1+ and GFP− Thy1+ cells. While this initially results in a seemingly higher RGC yield, for the subsequent transplant analysis it must be kept in mind that only GFP-expressing donor RGCs can be traced.
Dynabeads mouse pan t
Manufactured by Thermo Fisher Scientific
Dynabeads Mouse Pan T is a magnetic bead-based product designed for the isolation and enrichment of mouse T cells from a variety of sample types. The product utilizes superparamagnetic beads coated with antibodies specific to mouse pan-T cell surface markers to capture and isolate T cells from cell suspensions.
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Lab products found in correlation
3 protocols using dynabeads mouse pan t
1
Thy1.2-based RGC Isolation Protocol
2
In Vivo T Cell Behavior Monitoring
3
In Vivo T Cell Behavior Monitoring
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Cells were isolated from lymph nodes and spleen of DsRed mice, sorted using Dynabeads Mouse Pan T (11443D, ThermoScientific) according to the manufacturer’s protocol and injected (5 × 106 T cells) retro-orbitally. Cell behavior was analyzed 16 hours later in MLN explants by 2-photon microscopy as reported41 (link), 42 (link), before and after FTY720-P.
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