Mice were retroorbitally injected with 2.5 μg of CD45.2 FITC (Biolegend, San Diego, CA) in 100 μl PBS. Three minutes later, mice were euthanized and spleens, lungs, and mediastinal lymph nodes (mLN) were aseptically isolated and processed as previously described [12 (link)]. Spleens and mLNs were passed through a 70 μM cell strainer to generate a single cell suspension. The lung was minced and then digested with Liberase (Roche, Indianapolis, IN). Red blood cells in the spleen and lung were lysed by ACK lysis buffer (Life Technologies, Carlsbad, CA). The total number of viable cells in each tissue was determined by trypan blue exclusion using a TC10 Automated Cell Counter (Bio-Rad, Hercules, CA).
Protocol detail
Rapid CD45.2 Labeling and Tissue Analysis
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Buffer
Cell
Fitc
Liberase
Lung
Lymph nodes
Mediastinal
Mice
Mlns
Red blood cells
Spleen
Tissue
Trypan blue
Corresponding Organization : National Institutes of Health
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Variable analysis
independent variables
- Retroorbital injection of 2.5 μg of CD45.2 FITC in 100 μl PBS
- Total number of viable cells in each tissue (spleen, lungs, and mediastinal lymph nodes (mLN))
- Time between injection and euthanasia (3 minutes)
- Tissue isolation and processing (aseptic isolation and processing as previously described [12])
- Tissue processing methods (passing spleens and mLNs through a 70 μM cell strainer, mincing and digesting lungs with Liberase, lysing red blood cells in spleen and lung using ACK lysis buffer)
- Viable cell counting method (trypan blue exclusion using a TC10 Automated Cell Counter)
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