Isolation of murine splenocytes
In order to study spleen cells (e.g. lymphocytes, granulocytes, other immune cells), it helps to make single-cell suspensions so that the cells can be manipulated ex vivo easily. This protocol suggests ways in which you can do this without a lot of equipment or expensive supplies.
All materials listed are for use with one mouse.
- 15ml conical tube
- 60mm petri dish
- 5ml pipet
- 100μm cell strainer (can substitute autoclaved fine nylon mesh for Protocol B)
- 3mL sterile disposable syringe, no needle attached (Protocol A only)
- frosted-end glass slides (x 2) (Protocol B only)
- 50ml conical tube (Protocol B only)
- 1L DMEM (with 4.5g/L glucose, L-glutamine, sodium pyruvate; from Mediatech, catalog# 10-013-CM)
- 100mL fetal bovine/calf serum
- 10mL 100x PSG (penicillin G sodium, streptomycin sulfate, L-glutamine; from Gibco, catalog# 10378-016)
- sterilize using 0.2μm filter; store at 4°
- ACK lysis buffer
- 1L deionized H2O
- 8.29g NH4Cl
- 1g KHCO3
- 37.2mg Na2EDTA
- pH solution to 7.2-7.4; sterilize using 0.2μm filter; store at 4°
- 70% ethanol
- 1x phosphate buffer solution
- trypan blue solution
- small plastic or glass beaker
- dissection stage (can be styrofoam shipping box lid wrapped in aluminum foil)
- P1000 pipette
- phase microscope
Note: If sterility is desired, then perform all steps within a laminar flow culture hood.
- Clean dissection stage with 70% ethanol.
- Add ethanol to the beaker and place ends of scissors and forceps into the beaker to sterilize.
- Add 8-10mL of DMEM-10 to the petri dish.
- Place the cell strainer into the dish with the DMEM-10.
- Wet fur on left side of sacrificed mouse using 70% ethanol.
- Cut out the spleen.
- Cut away the fur along the left side of the mouse, about half-way between the front and back legs.
- Cut open the body cavity.
- Remove the spleen using the forceps (the spleen is the color of a kidney bean; it is longer and flatter than the kidney).
- Place the spleen into the cell strainer. Using the plunger end of the syringe, mash the spleen through the cell strainer into the petri dish.
- Rinse the cell strainer with 5mL DMEM-10. Discard the strainer.
- Transfer the suspended cells to a 15mL conical.
- Spin cells at 800xg for 3 minutes.
- Discard supernatant and resuspend pellet in 1mL ACK lysis buffer. Incubate at RT for 5-10 minutes. Add 9mL DMEM-10 and spin as before.
- Discard supernatant and resuspend pellet in 3mL DMEM-10.
- Count cells (dilute 10μL cell suspension in trypan blue, and count with hemcytometer).
- same as Procedure A, except replace step 4 with:
- Sterilize the frosted end of the glass slides by dipping in or spraying with ethanol. Take care to only touch the non-frosted ends with your gloves.
- same as Procedure A, except replace steps 3 with:
- Place the spleen directly into the DMEM in the petri dish.
- Homogenize the spleen between the frosted ends of the slides.
- Pass the homogenized spleen through the cell strainer (or nylon mesh) mounted on a 50mL conical.
- Continue with step 4 of Procedure A.
- Keep cells on ice or at 4° if you do not plan to use them right away.
- If sterility is desired, perform all steps in a laminar flow culture hood.
Relevant papers and books
- Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells link (subscription required)