Computational protocol: A retinoic acid-dependent stroma-leukemia crosstalk promotes chronic lymphocytic leukemia progression

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Protocol publication

[…] Tissues were collected and fixed for 5 min in 4% (w/v) PFA (Sigma-Aldrich), then washed in PBS 1 × and dehydrated overnight in 30% sucrose (Sigma-Aldrich) at 4 °C. Samples were embedded in Tissue-Tek OCT compound (Bio-optica) and frozen in ethanol dry-ice bath (using Dehyol 95 (Bio-optica)). Around 8–10 μm thick sections were placed onto glass slides (Bio-optica), fixed in cold acetone for 5 min, dried, and kept at −80°C until used. Slides were incubated 30 min with a blocking solution of PBS at 0.5%FBS and 0.05% Tween (VWR) (PBS-T 0.05%), followed by primary (supplementary material) specific antibodies or secondary reagents (supplementary material). Primary antibodies, secondary antibodies, and streptavidin reagents were diluted in blocking solution PBS-T 0.05% and incubated for 1 h and 30 min, respectively. For anti-mouse and biotin-conjugated primary antibodies, additional incubation with MoM (Vector Lab) and Avidin/Biotin blocking solution (Vector Lab), respectively, were performed following the manufacturer’s instructions. Nuclei were visualized with DAPI (Fluka), and mounting was performed with Mowiol (Calbiochem). For detection of MAdCAM-1, CXCL13 and PDPN antibodies, Tyramide Signal Amplification kit (Perkin Elmer) was used. To visualize proliferating cells, Click-iT® EdU Imaging Kits (Invitrogen) was employed accordingly to manufacturer’s protocol. Confocal images were acquired using Leica TCS SP2 and Leica TCS SP8 microscopes. Digital images were recorded in separately scanned channels with no overlap in detection of emissions from the respective fluorochromes. Final image processing was performed with Adobe Illustrator CS4 and Adobe Photoshop CS4. For immunohistochemistry of human biopsies, tissue sections from formalin-fixed, paraffin-embedded blocks were used. Sections were stained with primary goat polyclonal antibody to CXCL13 (dilution 1:30; R&D Systems) and upon appropriate antigen retrieval, reactivity was revealed using biotinilated anti-goat IgG (dilution 1:250; Vector Laboratories) followed by 3,3′-diaminobenizidine tetrahydrochloride (DAB), and finally counterstained with H&E according to standard protocols. After dehydratation slides were permanently mounted in Eukitt (Bioγ-Optica). Digital images were acquired using the Olympus BX60 microscope with DP-70 Olympus digital camera and processed using Analysis Image Processing software. [...] Eμ-TCL1, transplanted and control mouse tissues (peripheral blood, lymph nodes, omentum, mesentery, and femoral bone marrow) were collected from mice either alive (peripheral blood) or killed (other tissues). Solid tissues were smashed and filtered on a 40 μm cell strainer (Corning). Single-cell suspensions were washed in PBS and erythrocytes were depleted using an ammonium chloride solution (ACK) lysis buffer (NH4Cl 0.15 M, KHCO3 10 mM, Na2EDTA 0.1 mM, pH 7.4). For flow cytometry analyses, samples were incubated for 15 min at RT with Mouse BD Fc Block™ (purified rat anti-mouse CD16/CD32; BD Bioscience Pharmingen). Then cells were washed and incubated for 15 min at 4 °C with conjugated antibodies and finally data were acquired on FACSCanto™ II (BD Biosciences) and analyzed using FlowJo software (Tree Star). For culture experiments, B-lymphocytes were collected from the spleen, purified and enriched by negative depletion (EasySep™ Mouse B Cell Enrichment Kit; StemCell Technologies). The purity of all murine CLL samples and control B cells was always more than 90%. Human CLL cells were purified immediately after blood withdrawal, by negative depletion using the RosetteSep B-lymphocyte enrichment kit (StemCell Technologies). The purity of all human preparations was always more than 99% and the cells co-expressed CD19 and CD5 on their cell surface as checked by flow cytometry (FC500; Beckman Coulter); preparations were virtually devoid of natural killer cells, T lymphocytes, and monocytes. Human primary samples were obtained from RAI stage 0-1 CLL patients, after informed consent as approved by the Institutional committee (protocol ViVi-CLL) of San Raffaele Scientific Institute (Milan, Italy) in accordance with the Declaration of Helsinki. […]

Pipeline specifications

Software tools Adobe Illustrator, FlowJo
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus, Homo sapiens
Diseases Leukemia, Neoplasms, Leukemia, Lymphocytic, Chronic, B-Cell
Chemicals Retinoids, Tretinoin