Glomeruli Isolation: A Technique to Obtain Intact Glomeruli from Murine Kidney

In vertebrate kidneys, the nephrons consist of multiple glomeruli - a dense network of blood vessels - which play a primary role in the filtration of waste from the body.

To isolate glomeruli, begin by taking a freshly dissected mouse kidney. Use forceps to peel off the renal capsule from the kidney and place the clean kidney in a new culture dish. Now, position the dish over ice to avoid kidney degradation. Mince the kidney into small pieces.

Next, resuspend the minced kidney pieces in a suitable buffer solution. Filter the tissue suspension through a large-sized mesh filter and collect the flowthrough in a fresh dish. Repeat this step a few more times while progressively decreasing mesh size every time, till glomeruli dissociate from the kidney fragments.

Finally, wash the filter with the desired culture media to collect the smaller entrapped glomeruli while the kidney fragments remain in the filtrate. Transfer the glomeruli onto an ultra-low attachment plate. Visualize the plate using an inverted light microscope.

With the help of a micropipette, capture a single glomerulus and transfer it to a fresh well. Supplement the well with culture media and use the isolated glomerulus for further analysis.

After freeing the kidneys as outlined in the text manuscript, hold the kidney with the surgical forceps and use another pair of forceps to pull off the renal capsules. After this, place the kidneys into the wells of a 6-well culture plate that each contain 2 milliliters of HBSS and place the culture plate on ice.

Next, transfer the kidneys to a 100-millimeter Petri dish and use two scalpels to mince them into small pieces that are approximately 1 to 2 millimeters. Keep the minced kidney pieces wet with HBSS.

Next, place the kidney pieces on top of a 300-micrometer metal sieve and press the kidney through the sieve using the plunger from a 20-milliliter syringe. Repeatedly rinse the sieve with HBSS in between and use a serological pipette to collect the flow-through and transfer it to a clean Petri dish.

Use a scalpel to scrape off everything that remains in the bottom of the sieve and transfer it to the collected kidney homogenate. Rinse the kidney homogenate through a 75- and a 53-micrometer sieve with HBSS. Then, wash both sieves with HBSS to remove all of the smaller structures.

Collect the kidney structures and material that remain in the sieves by washing the upper surface of each with DMEM supplemented with 20% fetal calf serum and transfer the material to the wells of a 6-well ultra-low attachment microplate.

Bring the ultra-low attachment microplate to an inverted light microscope and use a 20-microliter pipette to collect single encapsulated and/or decapsulated glomeruli.