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

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).
11. Three days later, add another 10 ml of RPMI 10%FBS with 20 ng/l of rmGM-CSF to each of the prepared plates.
12. Three days later 10 ml of cell suspension are recovered from each Petri dish, centrifuged as in 1.6, resuspended in the same volume of RPMI 10% FBS with rmGM-CSF and returned to the Petri dish. Cells are cultured for 2 additional days in the CO₂ incubator.

2. Collection and labeling of DCs

1. After 8 days in culture loosely adherent cells are recovered by washing the Petri dishes with fresh medium. This protocol renders around 2-4 x10⁸ DCs in our hands. Cells can be analyzed for DC phenotype by flow cytometry.
2. Collected cells are then labeled with the Qtracker 655 Cell Labeling Kit strictly following the manufacturer’s instructions.
3. We got excellent results labeling murine bone marrow-derived DCs with Qdots at a 10 nM concentration. To accomplish this, aliquots of Qtracker Cell Labeling Kit components A and B are mixed in equal volumes, incubated for 5 min at room temperature, and immediately diluted 1/100 in fresh medium with vortexing for 30 s.
4. To stain 5 x 10⁶ DCs, mix 5 μl of each kit components A and B in an eppendorf tube and incubate for 5 min at room temp. Then, add 1 ml of RPMI 10% FBS incubate, and vortex for 30 second.
5. Add 0.5 ml of cell suspension containing 5 x10⁶ DCs, and incubate at 37°C for 60 min.
6. Then, wash cells 2X in RPMI 10% FBS (1,100 RPM). Cells can be resuspended in RPMI for further culture or in PBS for injection in experimental mice.

3. Evaluation of labeled DC

1. Cell labeling can be evaluated by means of fluorescent microscopy. To accomplish this, a drop of cell suspension is added to a microscope slide, covered with a glass coverslip, and evaluated with a fluorescent microscope taking into account that these Qdots have emission and excitation spectra of 565nm and 405–525nm respectively (Fig. 3)
2. At this point, the labeled cells can be used to inject animals, or maturation can be induced by incubating these cells for 48 h (37°C, 5 CO₂) in the presence of LPS (100 ng/ml) and TNF- (20 ng/ml). Fluorescent labeling is not affected by DC maturation (Fig. 4).

4. Representative Results:

Herewith we described how to prepare murine bone marrow-derived DCs and how labeled them with fluorescent Qdots. Fig. 1 summarizes the procedure to obtain the cell, while Fig. 2 depicts the procedure to label them with Qdots. As shown in Fig. 3, almost all the cells are labeled by this procedure, and this is not affected by DC maturation with inflammatory factors (Fig. 4). Fig. 5 shows that Qdot-stained DCs (Fig.5a) behave similar to non-stained cells when treated with an inflammatory cocktail. Both cell preparations similarly upregulate expression of costimulatory molecules (Fig. 5b); and produce similar amounts of IL-6 (Fig. 5c) and nitric oxide (Fig. 5d) upon maturation stimuli. This agrees with previous published data indicating that Qdot staining does not affect the capability of DC to turn into mature cells capable of eliciting immune responses [6]. These cells can then be used for injecting experimental animals. As shown in Fig. 6, 2 days after intravenous injection of labeled DCs into mice, we were able to detect Qdot-stained cells in the spleen. This is also in agreement with previous published data showing the use of Qdot nanocrystals to evaluate migration of DC in vivo [6] by non-invasive imaging and flow cytometry.

Figure 1. Flow chart of DC generation from bone marrow. Herewith we depict the process to obtain bone marrow-derived DC. Both tibias and femurs are dissected and clean from surrounding tissue. Bone tips are preserved and the interior of the bones are flushed with medium. After eliminating red blood cells, bone marrow cells are cultured for 8 days in the presence of GM-CSF to differentiate them into DCs.

Figure 2. Flow chart of the labeling procedure. Equal aliquots of Qtracker Cell Labeling Kit components A and B are mixed in an eppendorf tube. Dendritic cells are collected from 8-day bone marrow cultures with GM-CSF and mixed with the labeling dye for 60 min at 37C. Then cells are washed in media to eliminate excess labeling particles.

Figure 3. Fluorescent microphotography of DCs immediately after labeling. A drop of labeled cells was deposited on a glass slide and covered by a coverslip. Then, samples were evaluated in a fluorescence microscope and images were acquired through a Micropublisher 5.0 Digital CCD Color Camera (Qimaging, Surrey, BC Canada).

Figure 4. Fluorescent microphotography of DCs after 48 h maturation in vitro. Labeled DCs were cultured for 48 h with LPS (100 ng/ml) and TNF-α (20 ng/ml) on glass slides on a CO₂ incubator (37°C, 5% CO₂). Then, samples were washed with PBS (5 min, 2X), fixed with acetone (15 min, 4°C) and counterstained with DAPI. Cells were evaluated in a fluorescent microscope as above.

Figure 5. Biological activity of Qdot stained mature DCs. Labeled DCs matured in vitro as above were analyzed by flow cytometry (A) and (B). (A) Qdot-stained cells give a strong signal in the FL3 (PercP) channel. Shaded histogram: unstained control. (B) Qdot-stained and unstained DCs were additionally stained with specific antibodies against maturation markers CD86 and CD40; and isotype controls. Cells were then analyzed in a FACSort flow cytometer (Becton Dickinson, San Jose, CA). Additionally, IL-6 (C) and nitric oxide (NO) were determined in supernatants of mature Qdot-stained DCs and controls. Levels of IL-6 and nitric oxide were determined by means of ELISA analysis and Griess assay as we have previously described ^{7, 8}. All the data showed in this figure is representative of two or three independent experiments showing similar results. Data was analyzed by ANOVA by using the GraphPad software.

Figure 6. Detection of labeled DCs in dissected tissues. Labeled mature DCs (1×10⁶ cells) were intravenously injected into C57BL/6 mice. (A) Two days later mice were sacrificed and spleens collected, snap frozen, embedded in OCT, and 8 μM sections prepared using a cryostat. Then, samples were fixed with acetone (15 min, 4°C) and counterstained with DAPI. Cells were evaluated in a fluorescent microscope as above. Fig 6a: 40X magnification, Fig 6b: 100X magnification, Fig. 6C, 400X magnification.