Dendritic cell isolation experiments

Summary

This unit describes two methods of isolating dendritic cells (DCs): isolating DCs from mouse spleen and obtaining large numbers of DCs from mouse bone marrow precursor cells.

Author: J.E. Collier et al, Translator: Xuitao Cao et al. This experiment is from the "Comprehensive Immunology Laboratory Guide".

Operation method

Dendritic cell isolation experiments

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BASIC PROGRAM 1 Plastic adhesion and EA rose knot enrichment DC

This technique has been used for the separation of DC for many years with minor modifications (Steinman aZ., 1979).

Materials (see Appendix 1 for items with V)

Collagenase-digested spleen cell suspension (see Supporting Program)

High-density bovine serum albumin (BSA) solution

RPMI 1640 medium (e.g. Life Technologies), 4°C and 37°C

R P M I - 5 complete medium, 4°C and 37°C

Antibody-coupled sheep erythrocytes (EA)

Primary antibodies for flow cytometric analysis (Table 2. 3. 2)

Secondary antibodies for flow cytometry (synaptically conjugated mouse anti-rat I g G and I g M; e.g., BoehringerMannheim)

Beckman G H-3.7 and Sorvall HS-4 transcripts

15 ml and 50 ml polypropylene cone-bottomed tubes

Autoclaved 9in (~23c m) cotton and non-cotton filled pasteurized pipettes, 60m m diameter tissue culture dishes (e.g. Falcon)

1 . Centrifuge the collagenase-digested spleen cell suspension at 280 g for lOmin at 4°C and discard the supernatant. Rapidly resuspend the precipitate with high density BSA, approximately lml per spleen.

2 . Prepare BSA columns: Add 5 to 6 ml of cell suspension to a 15 ml centrifuge tube and carefully add 1.5 ml of RPMI-1640 medium at 4°C to the suspension to form a distinct interface. Prepare 4 or 5 BSA columns in this manner until the cell suspension has been fractionated.

3 . Centrifuge the BSA columns at 9500 g for 15 min at 4°C, accelerating slowly, taking care to turn off the "brake" switch.

4 . Carefully collect the cells at the interface with a cotton filled pasteurized pipette, aspirate all of the RPMI-1640 and the top l ml of BSA, and transfer the cells to another clean 50 ml centrifuge tube (discard the sediment). Fill the centrifuge tube with RPMI-1640 at 4°C to dilute the B S A and mix with slow shaking.

5. Centrifuge at 280 g at 4°C for 1 om m and discard the supernatant. Resuspend cells in 5 to 100 ml of RPMI-5 complete medium and place on ice.

6 . Count the viable cells by trypan blue staining (Appendix 3C, a spleen may yield IO7 low-density splenocytes).

7. Adjust the cell density to approximately ^IO7 cells/ml with RPMI-5 complete medium. Inoculate every 4 ml of suspension into a 60 m m dish until all cells are spread. Incubate for 90 miri to bond the DCs.

8 . Discard non-adherent cells: aspirate 37°C R P M I -1640 medium with a cotton filled pasteurized pipette and carefully wash the surface of the Petri dish to remove suspended cells until the surface of the dish changes from rough and cloudy to smooth and nearly clear. Repeat the wash once.

9 . Add 4 ml 37°C R P M I -1640 medium to each dish and incubate for 30 to 60 m i n to remove adherent lymphocytes . Repeat step 8 .

10. Observe the dishes under an inverted microscope using phase contrast. Most of the dark-colored star-shaped dendritic cells, some bright flat macrophages, and round cells such as lymphocytes and monocytes are visible. If the proportion of dendritic cells is not high, repeat steps 8 and 9.

11. Replace the liquid in each dish with 4 ml of clean RPMI-5 complete medium and incubate for 12 to 20 h.

12. Wash the dishes by aspirating 37°C RPMI-5 complete medium through a cotton filled pasteurized pipette. Transfer the washed cells into 15 ml centrifuge tubes on ice (each tube can contain 3 dishes of washed cells). Wash the dishes with 2 ml of RPMI-5 Complete Medium. Repeat this procedure until all the cells in the dishes have been collected.

13. Centrifuge the collected cells at 280 g for lOmin at 4°C and discard the supernatant. Resuspend the cells with I ml of dry RPMI-5 complete medium and place on ice.

14. Count the viable cells (0. ^5X106 ~ I.OX l0. ⁶ cells/spleen, about 80% for DC) by Taipan blue staining.

15. Add EA (to bind B cells and macrophages) in the amount of 50 EA (antibody-coupled sheep erythrocytes) per leukocyte and mix upside down. Centrifuge at 70 g for lOmin at 4°C. After centrifugation, place the tube on ice for 30 min without discarding the supernatant.

16. Dispose of part of the medium, leaving 2 to 2.5 ml. Use a cotton filled pasteurized pipette to gently blow the cell suspension 10 to 15 times to break up large clumps without destroying the rosette.

17. The resuspended cells are checked with a blood cell counter to confirm that each rosette is a white blood cell surrounded by red blood cells. If necessary, resuspend the cells again.

18. Prepare the BSA column: add 5 ml of high density BSA to a 15 ml centrifuge tube and carefully add 2.5 ml of rose bengal suspension to the top of the liquid to create a clear interface.
19-Centrifuge and collect cells as in steps 3 to 6 (2X 105 to 7X 105 cells/spleen, greater than 95% for DC).

20. Label the D C with the antibodies in Table 2. 3. 2 and check their purity by flow cytometry. For secondary antibodies, fluorescently conjugated mouse anti-rat Ig G and IgM were used.

Auxiliary protocols Collagenase digestion for preparation of spleen cell suspension

Collagenase-digested splenocyte suspensions yield 2 to 3 times the amount of DCs obtained by direct abrasion of the spleen by nondigestive methods (Unit 2.1). This method is also capable of loosening the DCs located at the periarterial sheath site.

Materials (see Appendix 1 for items with V)

4000 U/m l of collagenase D, dissolved on ice

H B S S, sterilized with Ca2+, M g 2+ (purchased from LifeTechnologies)

Mouse spleen

Hypodermic needle, 22 G X l M i n internal diameter (BectonDickinson)

10m l, 5m l - secondary syringe (e.g. Becton Dickinson)

IOOmm diameter Petri dishes (e.g. Falcon)

2 autoclaved serrated dissecting forceps (e.g. Roboz)

Autoclaved stainless steel mesh. -Cut 40 mesh stainless steel mesh into 5c m X 5c m squares, fold down the edges and bend the sides into a shallow rectangular bowl; wrap in tin foil and autoclave.

1. Dilute 2 tubes of Im l of 4000 U/m l of Collagenase D (enough for 20 spleens): Im l with 9 m l of H B S S (diluted to 400 U/m l, step 8 for use), Im l with 39 m l of H B S S (diluted to 100 U/ml). Place
on ice.

2 . Attach a 22 G needle to an I O m l syringe filled with l O O U / m l of collagenase. Add I O m l of the l O O U / m l solution to the I O O m m petri dish.

3 . Take another Petri dish of I O O m m diameter for the procedure. Hold the forceps in one hand, the syringe in the other, and the thumb
finger on the plunger. Hold the mouse spleen in the valvator and inject 100 U/m l of collagenase 100/xl by piercing the narrow edge of the splenic membrane with the needle. push the spleen against the needle with the forceps so that the needle advances a few millimeters. Repeat the collagenase injection , continuing until I m l of collagenase are injected into the spleen and the needle pierces the spleen from the other end.

4 . Tear open the spleen with the needle and transfer it to the collagenase-containing petri dish (step 2). Repeat until all spleens are injected and torn.

5 . Collect the cell suspension from the second petri dish and add to a 50m l centrifuge tube placed on ice. Rinse the petri dish with 3m l l 00U/ml collagenase and add to the above 50m l centrifuge tube.

6 . Add 3 ml l 00U /m l collagenase to the petri dish. Transfer the torn spleens sequentially to the petri dish. Using 2 forceps, tear them into small pieces with a side length of I m m so that they can pass through the inner diameter of a 5 m l pipette. When all the spleens have been shredded, add the collagenase solution from the petri dish where the spleen fragments were previously placed and blow vigorously several times with the 5 ml straw.

7 . Tilt the petri dish to remove the large pieces and transfer the cell suspension to the 50 m l centrifuge tube on ice from step 5. Wash the dish with a few milliliters of lOOU/m l collagenase and add to the centrifuge tube.

8 . Add IOml 400U/m l of collagenase to the spleen pieces left in the dish. Blow several times and leave in the incubator for 30 to 90 min.

9 . At the final stage of incubation, the fragments are blown vigorously and sucked onto a sterile steel mesh of a IOO m m diameter petri dish.

10. Secure the end of the stencil with forceps, wash the fragments with a few milliliters of lOOU/m l collagenase, and then crush the fragments with a sterile 5-ml syringe plunger. Mash until all red material is removed from the tissue. Wash the stencil again with a few milliliters of lOOU/ml collagenase.

11.Remove the stencil from the petri dish and discard the colorless adherent periplasmic debris. Blow vigorously on the liquid in the Petri dish and transfer it to the 50 m l centrifuge tube from step 7. Wash the dish with a few milliliters of collagenase and add to the centrifuge tube (about 0.5 % dendritic cells or more). 5 % dendritic cells or less).

12. If desired, centrifuge in high-density BSA (see Basic Plan 1).

BASIC OPTION 2 Cultivation of Dendritic Cells (DCs) from Mouse Bone Marrow Precursor Cells

This method (Inaba, 1992) overcomes the limited amount of "endogenous APCs" available in a tissue. Once cultured, large quantities of DCs can be used for cell biology research, genetic trait modification, and in vivo immunization. This approach is also important in studying the biology of DC differentiation.

Materials (

6 to 7 week old mice (preferably males, as they have larger bones; need to rest for at least 5 days after transportation; do not use stressed or dehydrated mice)

70 % ethanol

Ice-cold and room temperature RPMI-1640 medium (e.g. Life Technologies)

Ammonium chloride solution 0-02mol/L Tris - C U p H 7. 2 (Appendix I) /0-14mol/L N H 4Cl

Antibody (hybridoma supernatant) for cleavage of lymphocytes (optional). For example, hybridoma R A 3-3A 1 (B 2 20 antibody; ATCC No. TIB146), G K 1.5 (C D 4 antibody; ATCC No. Tffi 207), 3.155
(CD 8 antibody; TIB211), M 5/114 (MHCI antibody; TIB120) - see Unit 1.3.

Rabbit Complement (Pel-Freez)

R P M I-5 complete medium

Mouse G M - C S F (m G M -C S F ) : can be derived from purified recombinant proteins or from conditioned medium of cell lines transfected with mouse G M - C S F gene (gift of Dr. A . Basil, Switzerland). (Gift of Dr. A. Lanzavecchia, Basel, Switzerland).

Antibodies for flow cytometric detection of DC: e.g., hybridoma supernatant of MHCII-like molecules (ATCC No. TIB120), hybridoma supernatant of B 7-2/C D 86 (Pharmingen), antibody to DEC-205 (ATCC No. HB 290).

Anatomical supplies

Sterile gauze pads

IOO m m Petri dish (e.g. Falcon)

3 ml syringe 25 G, 5/8in (1.58c m) needle

9in (23c m) pasteurized pipette (cotton filled and autoclaved)

Nytex Filter (3-40/26; Tetko)

15 ml and 50 ml polypropylene cone-bottomed tubes

2 4-well culture plates (e.g. Corning)

1 . Take mouse femurs and tibiae and keep them in RPMI-1640 medium on ice until all mice have been prepared. After removing the muscle from the bones (usually done on sterile gauze pads), the clean bones are placed in a new petri dish. The bones are then soaked in 70% ethanol for 2 min and finally washed twice with cold RPMI-1640 medium.

2. The ends (epiphyses) of the bone are subtracted with scissors and transferred to a separate petri dish. Use a syringe to aspirate 2 ml of R P M I-1640 medium to flush the marrow cavity to obtain marrow. Chop the epiphysis in another Petri dish. Mix the chopped epiphyses and the rinsed bone marrow from the marrow cavity together, break up the clumps with a pipette, pass the suspension through a sieve (to remove the particles) and collect in a 15 ml or 50 ml centrifuge tube.

3. Add 3 to Io ml of ammonium chloride solution to lysed erythrocytes. Allow to stand for 3 m i n at room temperature, then centrifuge at 280 g for IO min (1200 r/min Beckman GH-3.7 rotor) and discard the supernatant.

4 . Removal of lymphocytes (optional): Prepare the cells at I X l O ⁷ cells/m l with the appropriate concentration of hybridoma supernatant (usually 1:20 final concentration) and rabbit complement (usually 1:17 to 1:20 final concentration). The cells were incubated at 37°C in a water bath for lh per
20m i n vibration.

The authors used B 220 antibody (hybridoma R A 3-3A 1), C D 4 antibody (hybridoma G K 1.5), C D 8 antibody (hybridoma 3.155), and 1MCHII antibody (hybridoma M5/114).

5 . Bone marrow cells are washed twice with RPMI-1640 and centrifuged at 280 g for l0 min at room temperature. Live cells are counted (Appendix 30). Adjust the cell concentration with RPMI-5 complete medium to IX ^lO6 cells/ml.

6 . Add mouse recombinant GM-CSF to a final concentration of 700 to lOOOU/m l or 20 ng/ml. inoculate the cell suspension, which typically yields 4 X ¹⁰⁷ to 5 X ¹⁰⁷ bone marrow cells per mouse (without removal of lymphocytes), at Iml/well.
2 4-well plates.

7. Wash the cells every two days, removing the old medium each time, then tilt the 24-well plate and gently wash the cells with RPMI-1640 along the walls of the wells, then pipette out the wash solution and finally add I ml of new RPMI-5 complete medium containing 700~lOOOU/ml (~20ng/ml) mGM-CSF (Step 6).

8. Optional: Verification of the characterization of aggregates - MHC class II molecules are present in the intracellular compartments of immature DCs (MHC class II compartments or MIICs) By labeling with [3H]thymine, it is found that 10-20% of the cells are in the S-phase, and that the cells moderately express membrane MHC class II molecules, as analyzed by flow cytometry (Module 4,1 and Module 4,2), but that they do not express membrane MHC class II molecules, and that they do not express membranous MHC class II molecules. However
cells moderately expressed membrane M H C class II molecules, but not B 7-2/C D 86 co-stimulatory molecules. By day 6, the wells of the culture plate were filled with numerous aggregates or spheres of proliferating immature DCs.

9. Between days 5 and 8 (when enough aggregates have been produced), detach the aggregates from the adherent stromal cells by gently blowing with RPMI-1640 or RPMI-5 medium (aggregates will continue to be produced for several days thereafter). The detached cells were collected and centrifuged at room temperature, 280 g for lOmin, and the supernatant was discarded. Resuspend with R P M I -5 medium, highest
Resuspend in RPMI-5 medium at a maximum concentration of IXLO ⁶ cells/ml and spread in IOO m m diameter dishes, 10 ml per dish, to a maximum of IXLO ⁷ cells.

10. During the period of 24~48 h after cell transfer, the culture dish was shaken gently every 24 h to collect the non-adherent, non-proliferating and mature DCs, which were transferred to another collection tube for later study.

11. Count live cells (Appendix 30). Count live cells (Appendix 30.) using a flow cytometer (Module 4.1 and Module 4.2). The rate of DC acquisition is examined by counting large irregularly shaped cells with a flow cytometer (Module 4.1 and Module 4.2) or with a blood counting plate (Appendix 3C) (usually 5 X per animal).
(typically 5 X ^IO6 to ^IOXlO6 cells per animal, >60% expressing surface markers of mature DC).

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