Isolationofmurinesplenocytes

Overview

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. This protocol can also be used to make cell suspensions from other lymphoid organs, such as the thymus or lymph nodes (see Current Protocols in Immunology, Unit 1.9 [1]).

Materials

All materials listed are for use with one mouse.

Supplies

• 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)

Reagents

• DMEM-10 (about 20mL)

• sterilize using 0.2μm filter; store at 4°

• 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)

• ACK lysis buffer (1mL)

• pH solution to 7.2-7.4; sterilize using 0.2μm filter; store at 4°

• 1L deionized water

• 8.29g NH4Cl

• 1g KHCO3

• 37.2mg Na2-EDTA

• 70% ethanol

• trypan blue solution

Equipment

• scissors

• forceps

• small plastic or glass beaker

• dissection stage (can be styrofoam shipping box lid wrapped in aluminum foil)

• P1000 pipette

• hemacytometer

• phase microscope

• centrifuge

Procedure

Protocol A

Set-Up

1. Clean dissection stage with 70% ethanol.

2. Add ethanol to the beaker and place ends of scissors and forceps into the beaker to sterilize.

3. Add 8-10mL of DMEM-10 to the petri dish.

4. Place the cell strainer into the dish with the DMEM-10.

Procedure

1. Wet fur on left side of sacrificed mouse using 70% ethanol.

2. Cut out the spleen.

1. Cut away the fur along the left side of the mouse, about half-way between the front and back legs.

2. Cut open the body cavity.

3. Remove the spleen using the forceps (the spleen is the color of a kidney bean; it is longer and flatter than the kidney).

3. 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.

4. Rinse the cell strainer with 5mL DMEM-10. Discard the strainer.

5. Transfer the suspended cells to a 15mL conical.

6. Spin cells at 800xg for 3 minutes.

7. 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.

8. Discard supernatant and resuspend pellet in 3mL DMEM-10, discarding any dead cell mass.

9. Count cells (dilute 10μL cell suspension in trypan blue, and count with hemcytometer).

Protocol B

Set-Up

• same as Procedure B, except replace step 4 with:

1. 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.

Procedure

• same as Procedure B, except replace steps 3 with:

1. Place the spleen directly into the DMEM in the petri dish.

2. Homogenize the spleen between the frosted ends of the slides.

3. Pass the homogenized spleen through the cell strainer (or nylon mesh) mounted on a 50mL conical.

4. Continue with step 4 of Procedure A.

Notes

• 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.