Stale warning

This protocol has not been updated this year! Last update was on April 01, 2025.

Murine Splenocyte Isolation

Splenocytes is a blanket term for a large subset of cells that are present within the spleen (T-cells, B-cells, macrophages, fibroblasts, etc.). In this protocol, we are specifically interested in the macrophages. Additional purification methods, such as antibody or bead pull down, could be used to isolate specific cell types.

Splencoyte Dissection

Estimated time

15 minutes per mouse

  1. Sacrifice the animal.

  2. Generously spray the animal with 70% ethanol.

Note

If a MEF isolation is happening, allow the embyros to be isolated before proceeding to isolate the spleen.

  1. Make an incision in the abdomen of the animal and cut about half way up the animal.

  2. The spleen will be a long, slender, reddish organ on the left-side of the abdomen, below the stomach (Left-side here is from the point of view of the animal, if the animal is on the back for dissection, it will be on your right). The kidney will also be in this area, but it should have a rounder, bean-like shape.

  3. Excise the tissue and remove any additional tissue connected to the organ.

  4. Place the spleen into a petri dish with PBS until ready for downstream processing.

Note

If the downstream processing will be more than 10 minutes, keep the petri dish with the spleen on ice.

Splenocyte Isolation/Preparation:

Estimated time

30 minutes

  1. Place the spleen into a clean petri dish and using 2 razor blades, mince the spleen into a paste.

  2. Once the spleen has been thoroughly minced, add 5-10 mL of DMEM + 10% FBS to the dish

  3. Prime a 40 um strainer with 1 mL of DMEM + 10% FBS into a 50 mL conical tube

  4. Pipette up the suspension and pass through the primed strainer into the tube.

  5. Add additional DMEM + 10 % FBS to the dish to get up any remaining cell matter

  6. Using a pestle or the plunger of a syringe, mash the cell matter through the strainer

  7. Wash the strainer 2-3 times with 1 mL of DMEM + 10% FBS.

Note

If liquid is not moving through the strainer, carefully lift up the mesh strainer and using a P1000 with a clean tip, pull the media through the strainer and dispense into the conical tube

  1. Discard the strainer and syringe

  2. Centrifuge down the cell suspension at 1600 rpm for 5 minutes

  3. Aspirate the supernatant

  4. Resuspend the cell pellet in 2 mL of prewarmed RBC lysing solution

Note

I use 2 mL of RBC lysing solution per spleen

  1. Incubate the cells at 37C for 2 minutes

  2. Add 20-30 mL of PBS to the suspension and centrifuge at 1,600 rpm for 5 minutes

  3. Aspirate off the supernatant and resuspend the cells in DMEM + 10% FBS

Note

The cells will be very sticky here and hard to resuspend, but try to break up some of the clumps

  1. Prime another 70 um strainer and pass through the resuspended pellet. You will need to use a pestle here and wash the strainer at least 2-3 times.

Note

There will likely be a gooey mass that will not make it through the strainer and that is fine. This is likely most of the lysed RBCs

  1. At this point you can either directly count the cells via a hemacytometer or centrifuge down the cells again and resuspend into a small volume to plate

Note

Depending on the downstream processing steps the number of cells can differ greatly. I usually plate 1 spleen per 10 cm dish and then after 3-4 days, seed cells for my experiments.

Note

For culturing techniques and dissociation methods, take a look at the methods under bone marrow isolation