Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Summary

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Abstract

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches ^1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes ⁴.

Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Protocol

1. Dissection of mouse femurs and tibiae and culture of bone marrow cells

1. Sacrifice 2 mice by CO₂ asphyxiation and carefully dissect tibias and femurs without cutting the bone ends.
2. Clean the bones from all the attached tissues by using tissue paper. Be careful not to break the bones.
3. Sterilize the bones by immersion in 70% ethanol for 10 min in a 35 mm Petri dish. From this moment, work inside a biosafety hood to avoid contamination of the cell cultures.
4. Recover the bones from the ethanol and let them air dry for 5 min in a Petri dish inside the biosafety cabinet.
5. Cut the femurs in half, and the tibia by its thinnest tip. Infuse the inside of the bone with 1 ml of RPMI medium (without serum but with antibiotics) using a sterile syringe on a sterile Petri dish.
6. The cell suspension is collected and washed 2X in RPMI medium by 10 min centrifugation in a 15 ml centrifuge tube at 1,100 RPM in a refrigerated centrifuge (4°C) with a swinging bucket rotor.
7. After the last wash, resuspend the cells in 2 ml of ACK lysis buffer and incubate for 5 min at room temperature in order to eliminate red blood cells.
8. Add 13 ml of RPMI with 10% FBS, resuspend and wash 2X in this medium with the same settings as described in 1.6.
9. Count cells, adjust to 2 x 10⁵ cells/ml with RPMI 10% FBS, and add rm-GM-CSF (20 ng/ml final concentration) ⁵.
10. Add 10 ml of this suspension to a sterile, microbiological quality, 10 cm Petri dish, and culture in a CO₂ incubator (37°C, 5% CO₂).

Discussion

Murine myeloid) DCs have been extensively used in order to determine the efficacy and improvement of DC-based vaccines; investigate DC:T cell interactions or DC development; and determine their role in various diseases ^9-11. Herewith we show how to generate DCs from precursors recovered from bone marrows of tibias and femurs. We recover the bones without cutting the tips, allowing us to sterilize them by submersion in ethanol 70%, thus reducing the probability of contamination. To differentiate DCs from bone marrow cells we only use GM-CSF as previously described ⁵. Although some protocols also use IL-4, it has been reported that this cytokine is not necessary when working with high levels of GM-CSF ¹². Indeed, we have previously demonstrated that these DCs are able to induce immune responses ¹³. Also, care has to be taken to recover only loosely adherent cells from 8-day cultures by washing the Petri dishes with medium since attached cells show a more monocyte-like phenotype. Here we show the labeling of DCs with fluorescent Qdot particles. This labeling has some advantages respect to other methods. First, the Qdots particles are easily incorporated into the cells. Second, the fluorescent signal is very high and is not altered by DC maturation. Third, the fluorescence is not lost when cells or tissues are fixed with solvents such as acetone, contrary to what happens if GFP is used to tag DCs ¹⁴, giving more flexibility at the moment to choose staining protocols. Finally, the high fluorescent signal given by these particles allows visualization of the cells despite tissue auto-fluorescence. As previously described ⁶, Qdot staining did not affect the maturation capability of these cells. Herewith we show that Qdot-stained DCs behave in a similar way as non-stained DCs, upregulating costimulatory molecules, and producing IL-6 and nitric oxide in response to inflammatory stimuli. Although DCs are cells specialized in triggering immune responses, they have been shown to participate in pathological conditions such as cancer and atherosclerosis ^{4, 15, 16}. They have been also claimed to participate in angiogenic process ^{17, 18}, even suggested as structurally participating in the developing of new vessels ^{19, 20}. Thus, methods that allow for DC tracking in vivo, and determining their geographical localization in different tissues ^{4, 21, 22} are very valuable.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6 mice	Jackson Laboratory		Females, 6-8 weeks old
RPMI	Invitrogen	11875-119
Fetal bovine serum, qualified	Invitrogen	10437-028
Antibiotic-antimycotic	Invitrogen	15240-096
PBS	Invitrogen	10010-049
ACK Lysing Buffer	Lonza Inc.	10-548E
Recombinant murine GM-CSF	PeproTech Inc	315-03
Qtracker 655 Cell Labeling Kit	Invitrogen	Q25021MP
Lipopolysaccharide	Invitrogen	tlrl-eblps
Recombinant murine TNF alpha	PeproTech Inc	315-01A
CD86 antibody	BD Biosciences	553691
CD40 antibody	BD Biosciences	553791
Griess reagent system	Promega Corp.	G2930
IL-6 capture antibody	eBioscience	13-7061-81
IL-6 detection antibody	eBioscience	13-7062-81