Isolation of mouse spleen dendritic cells - collagenase digestion to prepare spleen cell suspension

Auxiliary program:

Preparation of spleen cell suspension by collagenase digestion

Experimental Materials:

1. 4000U/ml collagenase D, melted and placed on ice

2. HBSS, sterilized, containing Ca ⁺ , Mg ²⁺ (purchased in Life Technologies)

3. Mouse spleen

4. Hypodermic needle, 22G × 1 ^1/2 in diameter (Becton Dickinson)

5. 10ml, 5ml disposable syringe (such as Becton Dickinson)

6. 100mm diameter petri dish (eg Falcon)

7. Sawtooth anatomy of 2 autoclaves (eg Roboz)

8. Autoclaved stainless steel mesh: Cut a 40-mesh stainless steel mesh into small squares of 5cm × 5cm, fold the edges down, and then bend the four sides into a shallow rectangular bowl; the foil is wrapped and autoclaved.

experimental method:

1. Dilute 2 tubes of 1 ml of 4000 U/ml collagenase D (sufficient for 20 spleens): 1 ml of 9 ml HBSS (diluted to 4000 U/ml, step 8), 1 ml of 39 ml of HBSS (diluted to 100 U/) Ml). Place on ice.

2. Attach the 22G needle to a 10 ml syringe filled with 100 U/ml collagenase. 10 ml of 100 U/ml solution was added to a 100 mm Petri dish.

3. Take another 100mm diameter petri dish for operation. Hold the forceps in one hand, the syringe in the other hand, and the thumb on the piston. The spleen of the mouse was clamped with forceps, and the narrow edge of the spleen capsule was pierced with a needle, and 100 ml of 100 U/ml collagenase was injected. Use a forceps to push the spleen toward the needle and advance the needle a few millimeters. The collagenase was repeatedly injected and continued until 1 ml of collagenase was injected into the spleen, and the needle pierced the spleen from the other end.

4. Use a needle to tear the spleen and transfer it to a Petri dish containing collagenase (Step 2). Repeat until all spleens are injected and torn.

5. Collect the cell suspension from the second dish and add to a 50 ml centrifuge tube placed on ice. The dish was rinsed with 3 ml of 100 U/ml collagenase and added to the above 50 ml centrifuge tube.

6. Add 3 ml of 100 U/ml collagenase to the Petri dish. The shredded spleen was transferred to a Petri dish in turn. They were torn with 2 tweezers into small pieces of 1 mm on the side so that they could pass through the inner diameter of the 5 ml pipette. After all the spleens were shredded, the collagenase solution in the petri dish previously placed on the spleen pieces was added and violently blown several times with a 5 ml pipette.

7. Incline the Petri dish, remove the large pieces of debris, and transfer the cell suspension to the 50 ml centrifuge tube placed on ice in step 5. The dish was washed with a few milliliters of 100 U/ml collagenase and added to a centrifuge tube.

8. Add 10 ml of 400 U/ml collagenase to the spleen pieces left in the dish. After pipetting several times, place in the incubator for 30-90 minutes.

9. In the final stage of the incubation, smash the pieces and suck them onto a sterile steel mesh of 100 mm diameter Petri dishes.

10. Secure one end of the stencil with tweezers, wash the pieces with a few milliliters of 100 U/ml collagenase, and then grind the pieces with a sterile 5 ml syringe plunger. Smash until all red material is removed from the tissue. The steel mesh was washed with a few milliliters of 100 U/ml collagenase.

11. Remove the stencil from the Petri dish and discard the colorless adherent granules. The liquid in the Petri dish is violently blown and transferred to the 50 ml centrifuge tube of step 7. The dish was washed with a few milliliters of 100 U/ml collagenase and added to a centrifuge tube (about 0.5% dendritic cells or less).

12. If necessary, centrifuge with high density BSA.

appendix:

Antibody-conjugated sheep red blood cells (EA):

2.5 ml of collected sheep erythrocytes (extracted in Alsever solution; Cocalico) were washed 3 times with PBS, and centrifuged at 350 g for 5 min after each washing. It was then diluted to 5% cell suspension (10 ⁹ cells/ml) with fresh PBS. Highly sensitized rabbit anti-erythrocyte serum was diluted 1 :50 in a 5% cell suspension (possibly forming a subagglutinated dose of antibody). Incubate for 2 h at room temperature and occasionally gently invert the solution. The formed EA was washed 3 times with RPMI 1640 (Life Technologies), and then EA was diluted to 5% cell suspension with PBS. Store at 4 ° C for 2 weeks and wash once with RPMI before use.

RPMI-5 complete medium:

RPMI1640 medium (Life Technologies), containing:

5% FBS, heat inactivated at 56 ° C for 1 h

10mmol/L HEPES

20mg/ml gentamicin sulfate (Life Technologies)

50mmol/ml 2-ME

Filtration sterilization

High density BSA solution:

In a 1 L volumetric flask, add 186 ml PBS, 29 ml 1 mol/L NaOH, 65 ml water (be careful, pay attention to the liquid and do not splash inside the bottle). Do not stir, apply 106g BSA (Cohn fraction V, recommended full BSA) on the surface of the solution, cover with tin foil, and chill overnight to dissolve BSA slowly.

The next day, gentle shaking has turned into a clear, slightly brown solution; the refractive index is measured to the nearest fourth decimal place. The correct density of BSA (1.080 g/ml) has a refractive index of 1.384 to 1.385 at 25 °C. After correcting the refractive index, the pH was measured with a test paper. The pH should be between 7.0 and 7.4 and generally no adjustment is required.

Sterile filtration solution. A 47 mm filter (Millpore type) was placed on top of a 500 ml filter unit (Nalgene #156-4045) and the filter was wetted with a small amount of BSA. Vacuum filter for a few minutes until the BSA is filtered out. Remove the vacuum pump and carefully add the remaining BSA solution to the reservoir in the upper part of the filter (avoid foaming. Use a vacuum pump and filter to filter the remaining BSA, ≥ 10 min). It can be stored for 3 months at 4 °C.