of Central Nervous System (CNS) Infiltrating Cells

Fig. 1

Isolation of CNS infiltrating cells from C57Bl/6 mice with EAE. (a) Absolute numbers of live and CD90 positive cells. Data represents CNS isolated material from 19 EAE diseased mice, with clinical scores ranging from 3.5 to 4.5, from 7 independent experiments. (b) Surface markers expression profile of CNS infiltrating lymphocytes (CD45+CD11b−), macrophages and microglia represents greater amounts of inflammatory cells in EAE diseased mouse (score 4) compare to nondiseased mouse (score 0). (c) Cytokine expression profile of CNS infiltrated CD4+CD40L+ T cells isolated from a mouse with clinical score 4. Cells were stimulated ex vivo in the presence of MOG peptide and Brefeldin A for 6 h

2 Materials

Prepare all equipment and materials in advance, as the time is critical for cell survival during the isolation procedure. Only digestion solution and Percoll fractions should be prepared shortly before usage.

2.1 Equipment

Surgical instruments: scissors, forceps, i.v. perfusion system, razor blades.

Suppliers: 100 mm petri dishes, 15 ml Falcon tubes, 5 ml and 10 ml medical syringes, hypodermic needles (BD): 0.9 × 40 mm 20 G “yellow,” 1.2 × 40 mm 18 G “red,” 0.9 × 70 mm 20 G “long yellow” (see Note 1 ).

Equipment: vortexer, centrifuge with swing-out bucket rotor, water bath.

2.2 Reagents

Mouse anesthesia: Isofluran (see Note 2 ).

70 % Ethanol.

Perfusion solution: Normal saline (0.9 % w/v NaCl) (see Note 3 ).

Phosphate buffered saline (PBS), without Calcium and Magnesium.

PBS, with Calcium and Magnesium.

10×PBS, without Calcium and Magnesium.

Hanks’ Balanced Salt Solution (HBSS), without Calcium and Magnesium (see Note 4 ).

Fetal calf serum (FCS).

Cell isolation/storage buffer (PBS/FCS): PBS supplemented with 2 % FCS.

10.

Digestion solution: PBS with Calcium and Magnesium, 1.5 mg/ml Collagenase II, 50 μg/ml DNase I (see Note 5 ).

11.

Stock Isotonic Percoll (SIP) solution: 90 % Percoll plus 10 % 10×PBS, without Calcium and Magnesium (see Note 6 ).

12.

30 %—Percoll fraction: 30 % SIP plus 70 % PBS, without Calcium and Magnesium.

13.

37 %—Percoll fraction: 37 % SIP plus 63 % HBSS, without Calcium and Magnesium.

14.

70 %—Percoll fraction: 70 % SIP plus 30 % PBS, without Calcium and Magnesium (see Note 7 ).

3 Methods

Keep biological material on ice unless otherwise specified. The stated volumes are calculated for the whole CNS, i.e. brain plus spinal cord of one mouse. Cell isolation from separated CNS parts is possible with reduced volumes. If further culturing of the cells is required, the isolation should be performed under sterile conditions, e.g. with sterile instruments, suppliers, solutions and under a laminar flow cabinet (see Note 8 ).

3.1 CNS Tissue Isolation and Dissociation

Deeply anesthetize mouse, make sure there is no pain reflex (see Note 9 ).

Spray mouse with ethanol, cut the skin, and open thoracic cavity. Make a small cut in the right atrium of the heart and insert the perfusion needle 0.9 × 40 mm into the left cardiac ventricle (see Note 10 ).

Run 40–50 ml of the perfusion solution for about 3–5 min through cannula with low drip until the effluent solution from the right atrium doesn’t consist of blood, i.e. become fully transparent (see Note 11 ).

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