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

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, put on ice after thawing
2. HBSS, sterilized, containing Ca+, Mg2+
3. Mouse spleen
4. Hypodermic injection needle, 22G×11/2 in inner diameter
5. 10ml, 5ml disposable syringe
6. 100mm diameter petri dish
7. 2 serrated dissecting forceps in an autoclave
8. Autoclaved stainless steel mesh: Cut the 40-mesh stainless steel mesh into small squares of 5cm×5cm, fold down the edges, and then bend the four sides into a shallow rectangular bowl; wrap it with foil and autoclave.
experimental method:
1. Dilute 2 tubes of 1ml 4000U/ml Collagenase D (enough for 20 spleens): 1ml add 9ml HBSS (dilute to 4000U/ml, use in step 8), 1ml add 39ml HBSS (dilute to 100 U/ ml). Place on ice.
2. Connect the 22G needle to a 10ml syringe and fill it with 100 U/ml collagenase. Add 10ml of 100 U/ml solution to a 100mm petri dish.
3. Take another petri dish with a diameter of 100mm for operation. Hold the tweezers in one hand, the syringe in the other hand, and place your thumb on the piston. Clamp the mouse spleen with tweezers, pierce the narrow edge of the spleen capsule with a needle, and inject 100 U/ml collagenase 100ml. Use forceps to push the spleen toward the needle, advancing the needle a few millimeters. Repeat the injection of collagenase until 1ml of collagenase is injected into the spleen, and the needle pierces the spleen from the other end.
4. Tear open the spleen with a needle and transfer it to a Petri dish containing collagenase (step 2). Repeat the operation until all the spleens are injected and torn apart.
5. Collect the cell suspension from the second petri dish and add it to a 50ml centrifuge tube on ice. Rinse the petri dish with 3ml of 100 U/ml collagenase and add it to the above 50ml centrifuge tube.
6. Add 3ml of 100 U/ml collagenase to the petri dish. Transfer the shredded spleen to a petri dish in turn. Use 2 tweezers to tear them into small pieces with a side length of 1mm so that they can pass through the inner diameter of a 5ml straw. When all the spleens have been shredded, add the collagenase solution in the petri dish where the spleen fragments were previously placed, and blow vigorously with a 5ml pipette several times.
7. Tilt the petri dish to remove any large pieces of debris, and transfer the cell suspension to a 50ml centrifuge tube on ice in step 5. Wash the petri dish with a few milliliters of 100 U/ml collagenase and add it to the centrifuge tube.
8. Add 10ml 400 U/ml collagenase to the spleen fragments left in the petri dish. Place in an incubator for 30-90 minutes after pipetting several times.
9. At the final stage of incubation, blow the fragments vigorously and suck them onto a sterile steel net with a diameter of 100 mm in a petri dish.
10. Fix one end of the steel mesh with tweezers, wash the fragments with a few milliliters of 100 U/ml collagenase, and then grind the fragments with a sterile 5ml syringe plunger. Smash until all the red material is removed from the tissue. Use a few milliliters of 100 U/ml collagenase to wash the steel mesh.
11. Remove the steel mesh from the petri dish, and discard the colorless and adhered envelope fragments. Vigorously pipette the liquid in the petri dish and transfer it to the 50ml centrifuge tube in step 7. Wash the culture dish with a few milliliters of 100 U/ml collagenase and add them to the centrifuge tube (about 0.5% dendritic cells or less).
12. If necessary, use high-density BSA centrifugation.
appendix:
Antibody-conjugated sheep red blood cells (EA):
The collected sheep red blood cells (extracted from Alsever solution; Cocalico) were washed with PBS for 3 times, and centrifuged at 350g for 5min after each wash. Then dilute with fresh PBS to a 5% cell suspension (109 cells/ml). Dilute the highly sensitized rabbit anti-erythrocyte serum in a 5% cell suspension at a ratio of 1:50 (may form a sub-agglutinated dose of antibody).
Incubate at room temperature for 2 hours, gently inverting the solution occasionally. The formed EA was washed 3 times with RPMI1640 (Life Technologies), and then the EA was diluted to a 5% cell suspension with PBS. Store at 4°C for 2 weeks, and wash again with RPMI before use.
In a 1L volumetric flask, add 186ml PBS, 29ml 1mol/L NaOH, 65ml water (operate carefully, be careful not to splash the liquid in the bottle). Without stirring, spread 106g of BSA on the surface of the solution, cover it with tin foil, and refrigerate overnight to slowly dissolve the BSA.
The next day, gentle shaking has turned into a clear, brownish solution; measure the refractive index to the fourth decimal place. At 25°C, the refractive index of BSA (1.080g/ml) with the correct density is 1.384-1.385. After correcting the refractive index, test the pH with a test paper. The pH should be 7.0-7.4, generally no adjustment is required.
Sterile filter the solution. Place the 47mm filter membrane on top of a 500ml filter device, and wet the filter membrane with a small amount of BSA. Vacuum filtration for several minutes, until the BSA is filtered out. Remove the vacuum pump and carefully add the remaining BSA solution to the reservoir above the filter (avoid foaming. Use the vacuum pump and filter to filter the remaining BSA, ≥10min). It can be stored for 3 months at 4°C.