Sampling and Visualization: Blood and Organ Concepts in Mice, Day 49

Mouse organ and blood sampling are foundational for immunology and preclinical research. Blood can be collected via 𝘳𝘦𝘵𝘳𝘰-𝘰𝘳𝘣𝘪𝘵𝘢𝘭 𝘣𝘭𝘦𝘦𝘥𝘪𝘯𝘨, 𝘵𝘢𝘪𝘭 𝘷𝘦𝘪𝘯, 𝘰𝘳 𝘤𝘢𝘳𝘥𝘪𝘢𝘤 𝘱𝘶𝘯𝘤𝘵𝘶𝘳e, depending on sample volume and downstream analysis [1]. Post-euthanasia, immune organs like spleen, thymus, lymph nodes, and bone marrow are harvested for cellular and molecular assays.  

𝗧 𝗖𝗲𝗹𝗹 𝗜𝘀𝗼𝗹𝗮𝘁𝗶𝗼𝗻: 
• Spleens or lymph nodes are mechanically dissociated and filtered to create single-cell suspensions. 
• Red blood cells are lysed using ammonium-chloride-potassium (ACK) buffer. 
• Negative selection using magnetic beads removes unwanted cells, leaving untouched CD3⁺ T cells for downstream assays such as flow cytometry, ELISpot, or activation assays [2]. 
• Positive selection can also be used, but may activate T cells.   

𝗕 𝗖𝗲𝗹𝗹 𝗜𝘀𝗼𝗹𝗮𝘁𝗶𝗼𝗻: 
• Spleen or peritoneal cavity cells are similarly prepared. 
• Magnetic beads target CD19⁺ or B220⁺ populations, either via negative or positive selection, depending on whether activation needs to be avoided [3]. 
 • Subsets such as B1 (peritoneal) or B2 (splenic) can be distinguished based on surface markers (CD5, CD11b). 

𝗠𝗮𝗰𝗿𝗼𝗽𝗵𝗮𝗴𝗲 𝗜𝘀𝗼𝗹𝗮𝘁𝗶𝗼𝗻: 
• Bone marrow-derived macrophages (BMDMs): Bone marrow cells are flushed from femur and tibia, plated in M-CSF-containing medium, and differentiated over 7 days [4]. 
• Peritoneal macrophages: Cells are harvested by peritoneal lavage, then plated to adhere, with non-adherent cells washed away. 
These cells can be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages using cytokine cocktails such as IFNγ/LPS or IL-4/IL-13.   

𝗔𝗻𝗲𝗰𝗱𝗼𝘁𝗲: 
During my master’s thesis, isolating a mouse aorta under a magnifier with fine pincettes made me feel an odd sense of pride, reminiscent of my father as a surgeon. While seemingly ´basic´, these manipulations instilled respect for precision and patience in biomedical research.  

𝗥𝗲𝗹𝗲𝘃𝗮𝗻𝗰𝗲 𝗳𝗼𝗿 𝗜𝗺𝗺𝘂𝗻𝗼𝗹𝗼𝗴𝘆 & 𝗖𝗔𝗥-𝗧: 
𝘏𝘪𝘨𝘩-𝘲𝘶𝘢𝘭𝘪𝘵𝘺 𝘪𝘴𝘰𝘭𝘢𝘵𝘪𝘰𝘯 is crucial for CAR-T expansion and cytokine profiling, as contaminated cells can skew functional assays and potency tests [5].  

𝗤𝘂𝗲𝘀𝘁𝗶𝗼𝗻 𝗳𝗼𝗿 𝘁𝗵𝗲 𝗮𝘂𝗱𝗶𝗲𝗻𝗰𝗲:  Your anecdotes from cell/organ isolation?  

Stay tuned for 𝗗𝗮𝘆 𝟱𝟬: 𝗠𝗮𝘀𝘁𝗲𝗿𝗶𝗻𝗴 𝗠𝗼𝘂𝘀𝗲 𝗛𝗮𝗻𝗱𝗹𝗶𝗻𝗴 

𝗥𝗲𝗳𝗲𝗿𝗲𝗻𝗰𝗲𝘀: 
1. PMCID: PMC3189662 
2. https://static.miltenyibiotec.com/asset/150655405641/document_7jrofkb68t5otc4h129j4c6i5p/IM0009079.PDF?content-disposition=inline 
3. DOI: 10.1016/j.cell.2024.03.038 
4. DOI: 10.1038/nri3073 
5. DOI: 10.1016/j.omtm.2023.101171 

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