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

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][2][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 macrophagecolony 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. Video LinkThe video component of this article can be found at https://www.jove.com/video/2785/ Protocol 1. Dissection of mouse femurs and tibiae and culture of bone marrow cells 1. Sacrifice 2 mice by CO 2 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 describe...

Long Isoform Mouse Selenoprotein P (Sepp1) Supplies Rat Myoblast L8 Cells with Selenium via Endocytosis Mediated by Heparin Binding Properties and Apolipoprotein E Receptor-2 (ApoER2)

“…Bone marrow cells were obtained from femur and tibia (21). Total RNA was isolated according to the manufacturer's protocol.…”

Assessment of Nanobiotechnology-Targeted siRNA Designed to inhibit NF-kappaB Classical and Alternative Signaling in Breast Tumor Macrophages

“…This effect can depend on the potency of the siRNA sequence itself, as different siRNA sequences against the same target gene can exhibit widely different abilities to recognize the targeted mRNA sequence for knockdown. 51 These results are significant because primary macrophages possess highly degradative phagocytic, endosomal, and lysosomal compartments, providing a formidable barrier to the cytosolic delivery of siRNA. 13 Moreover, because the ManNPs are internalized via an endocytotic receptor, these data indicate that ManNPs successfully mediate escape from the endosomal pathway, through a mechanism that is likely mediated by the pH-responsive, endosomolytic behavior of the core-forming terpolymer block.…”

“…Once the tumors reach a palpable size, macrophages labeled with Qtracker® 800 (Invitrogen) near infrared quantum dot cell tracking agent will be injected into the tumors at variable cell densities (3 different densities, n=4 mice per density), with PBS injections used as a control (n=3). 50,51 The animals will be imaged at 1, 6, 24, 48, 72, 96 hrs post injection using an IVIS imaging system to measure near infrared fluorescence as a measure of injected macrophage load retained in the tumor. experiments from Aim #1 and #2), the mice will be sacrificed and the lungs removed.…”

Antigen-loaded Dendritic Cell Migration: MR Imaging in a Pancreatic Carcinoma Model

“…Tumor growth was assessed as the product of perpendicular diameters measured with a caliper; tumor size was calculated by using the tumor size formula longest length 3 longest width (n = 6 mice with 12 tumors in each group) (27). The tumor sizes were measured at multiple time points after the administration of dendritic cells (1,5,10,15,20,25,30, and 35 days after tumor cell implantation). All mice were euthanized 35 days after dendritic cell or control injection.…”

“…Generation and Antigen Loading of Bone Marrow-derived Dendritic Cells Bone marrow-derived dendritic cells were prepared as previously described (20). Briefly, bone marrow cells were isolated from femur and tibia.…”