The overall goal of this procedure is to isolate lymph node stromal cells. This is accomplished by first disrupting the lymph node capsule. In the second step, the lymph node fragments are digested with collagenase four and D nase one.
The enzymes are then replaced with collagenase D and D nase one, and the fragments are mechanically disaggregated with an automated multichannel pipette. Ultimately, the cell surface molecule expression of the isolated lymph node cells can be characterized by flow cytometry. The main advantage of this technique over existing method is that with this method, the lymph node SHR cells are digested using a standardized enzymatic procedure complemented by mechanical disaggregation that preserves the viability and surface molecule expression of the cells.
Begin by placing the dissected lymph nodes in a sterile Petri dish containing two milliliters of ice cold medium. Then use two one milliliter syringes equipped with 25 gauge needles to disrupt the lymph node capsules. Next, transfer the disrupted tissue to a five milliliter conical tube containing 750 microliters of medium, supplemented with collagenase four and DNA one and a magnetic stir.
Then place the tube in a beaker containing preheated 37 degrees Celsius water on a magnetic stirrer and stir the cell slurries at one round per second for 30 minutes. After stirring, let the lymph node fragment settle and then carefully remove the supernatant. Now enriched for non stromal cells transfer to the lymph node fragments 750 microliters of fresh medium, supplemented with collagenase D and D nase one, and then stir the tissue for another five minutes at 37 degrees Celsius.
Next, use an automated multi-channel pipette to disaggregate the lymph node tissue fragments in a 700 microliter volume for 10 cycles at maximal speed, and then stir the tissue fragments again after 10 minutes. Further, disaggregate the tissue clumps with the automated multi-channel pipette for 99 cycles at maximal speed. Then add 7.5 microliters of 0.5 molar EDTA to the tube and mix the tissue suspension for another 99 cycles with the automated pipette.
After the last disaggregation cycle, add 750 microliters of medium to the cell solution and filter the cells through a 70 micrometer nylon mesh. Then pellet the cells for five minutes at 1500 Gs and four degrees Celsius. To visualize lymph node stromal cell populations by flow cytometry, stain the appropriate cell samples with live dead tracker and the antibodies of interest in 100 microliters of HBSS containing 2%FCS for at least 20 minutes at four degrees Celsius in the dark.
Then wash the cells in 500 microliters of HBSS with FCS and resuspend. The pellets in 100 microliters of HBSS and FCS run the cells on a flow cytometer gating out the CD 45 positive cells to exclude the hematopoietic cells, then gait on the live singlet population. Finally, plot GP 38 versus CD 31 to visualize the T-zone reticular cells, lymphatic endothelial cells, blood endothelial cells, and double negative cells.
Cell populations. The total cell number recovered post digestion of the lymph node stromal cell subsets was slightly higher in the just demonstrated protocol compared to the link and Fletcher protocols demonstrating that the viability of the lymph node stromal cells after isolation by this protocol is similar to that recovered using published protocols. T-zone reticular cells and lymphatic endothelial cells isolated via this and the link and Fletcher protocols were compared for IAB CD one 40 a CD 80, PD L one, and CD 40 expression.
The expression of all five surface molecules was higher upon digestion with the just demonstrated protocol and the link protocol for both stromal cell subsets, suggesting that the degradation of some surface molecules by collagenase four and D is less robust than with collagenase p and DYS space. After watching this video, you should have a good understanding of how to successfully isolate the four major subpopulation of lymph node stroma cells while preserving the expression of several surface molecules useful for their further characterization and analysis.
