Male Balb/c mice (8-10 weeks-old) were given foot pad injections in the right hind limb of 1×107 splenocytes from male DBA-2 mice (8-10 weeks-old). Recipient mice were immediately treated as follows (schematic in Figure 8A )
On day 4 after alloantigen challenge, the mice were sacrificed and the draining ipsilateral popliteal LNs (IL-pLNs) were collected. Following mechanical disruption of the pLNs, isolated cells were counted on a Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA, USA) to determine LN cellular expansion due to alloresponses. From each IL- pLN cell suspension, 1×106 cells were stained for flow cytometry using LIVE/DEAD fixable near infra-RED and antibodies against the following surface markers: anti-mouse CD3, anti-mouse CD8, anti-mouse CD62L (BD Bioscience), anti-mouse CD4 (e-Bioscience), anti-mouse CD44 and anti-mouse CD45 (Biolegend). Samples were evaluated using a Cytoflex flow cytometer and data analyzed with Flow Jo.
On day 4 after alloantigen challenge, the mice were sacrificed and the draining ipsilateral popliteal LNs (IL-pLNs) were collected. Following mechanical disruption of the pLNs, isolated cells were counted on a Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA, USA) to determine LN cellular expansion due to alloresponses. From each IL- pLN cell suspension, 1×106 cells were stained for flow cytometry using LIVE/DEAD fixable near infra-RED and antibodies against the following surface markers: anti-mouse CD3, anti-mouse CD8, anti-mouse CD62L (BD Bioscience), anti-mouse CD4 (e-Bioscience), anti-mouse CD44 and anti-mouse CD45 (Biolegend). Samples were evaluated using a Cytoflex flow cytometer and data analyzed with Flow Jo.