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Lympholite m

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Lympholite-M is a lab equipment used for the isolation and enrichment of mononuclear cells from whole blood samples. It is a specialized centrifugation device that separates different cell types based on their density.

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Lab products found in correlation

Lipopolysaccharides (lps), by Leica (2 mentions) Facsaria, by BD (2 mentions) Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions) Macs depletion column, by Miltenyi Biotec (1 mentions) Macs microbead, by Miltenyi Biotec (1 mentions) Midimacs separator, by Miltenyi Biotec (1 mentions) Enzyme free cell dissociation solution, by Thermo Fisher Scientific (1 mentions)

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Lympholite (7 citations)

5 protocols using lympholite m

1

Isolation of CD11b- and Sca-1-Positive Cells

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Ubiquitous red fluorescent mice (Ds-Red.T3, CD1-background) were sacrificed by cervical dislocation, spleens were explanted and mechanically minced. MNCs were isolated using a Ficoll gradient (Lympholite-M, Cedarlane). For preparation of CD11b- and Sca-1-positive cells, the same amount of spleen-derived MNCs was subjected to magnetic bead separation. In brief, spleen-derived MNCs were washed, re-suspended and mixed with colloidal superparamagnetic microbeads conjugated to CD11b or Sca-1 antibodies (MACS MicroBeads, Miltenyi Biotec). After incubation and additional washing, magnetic cell separation was performed by filling the cell suspension into a depletion column placed in the magnetic field of a magnetic bead separator (MACS Depletion Columns; MidiMACS Separator, Miltenyi). The collected effluent contained the negative MNC fraction depleted of CD11b- or Sca-1-positive cells, respectively. After taking the column out of the magnetic field, the attached CD11b-/Sca-1-positive MNCs were collected in buffer.
Andrié R.P., Beiert T., Knappe V., Linhart M., Stöckigt F., Klein A.M., Ghanem A., Lübkemeier I., Röll W., Nickenig G., Fleischmann B.K, & Schrickel J.W. (2019). Treatment with mononuclear cell populations improves post-infarction cardiac function but does not reduce arrhythmia susceptibility. PLoS ONE, 14(2), e0208301.
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2

Visualizing TCR-pMHC Interactions in Treg Cells

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CD4+ T cells were purified from AND-Tg Rag2−/− mice and stimulated by immobilized anti-CD3ε (145-2C11; 10 μg/ml) and anti-CD28 (PV-1; 1 μg/ml) in the presence of mouse recombinant IL-2 (10 ng/ml) and human recombinant TGFβ (5 ng/ml) for 3 d. The cells were retrovirally transduced with eGFP-tagged mouse PKC-θ or PKC-η for 24 h one day after the initial stimulation. On d 4 or later, the cells were sorted on a FACSAria (BD) to obtain homogenous population of highly GFP+ cells, which were maintained in culture. B cells purified from B10. BR mice and stimulated by LPS (Difco; 10 μg/ml) plus MCC (1 μM) were added to the cultured T cells for 1 d. Dead cells were removed by Lympholite-M (Cedarlane), and the CD4+ Treg cells were prestained by DyLight650-labeled anti-TCRβ (H57) Fab, conjugated with MCC-pulsed LPS-stimulated B cells for 5-10 min, fixed with 2% PFA, and imaged by confocal microscopy (Leica SP5) with ProLong gold antifade reagent with DAPI (Molecular Probes).
Kong K.F., Fu G., Zhang Y., Yokosuka T., Casas J., Canonigo-Balancio A.J., Becart S., Kim G., Yates JR I.I.I., Kronenberg M., Saito T., Gascoigne N.R, & Altman A. (2014). Protein Kinase C-η Controls CTLA-4-Mediated Regulatory T Cell Function. Nature immunology, 15(5), 465-472.
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3

Visualizing TCR-pMHC Interactions in Treg Cells

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CD4+ T cells were purified from AND-Tg Rag2−/− mice and stimulated by immobilized anti-CD3ε (145-2C11; 10 μg/ml) and anti-CD28 (PV-1; 1 μg/ml) in the presence of mouse recombinant IL-2 (10 ng/ml) and human recombinant TGFβ (5 ng/ml) for 3 d. The cells were retrovirally transduced with eGFP-tagged mouse PKC-θ or PKC-η for 24 h one day after the initial stimulation. On d 4 or later, the cells were sorted on a FACSAria (BD) to obtain homogenous population of highly GFP+ cells, which were maintained in culture. B cells purified from B10. BR mice and stimulated by LPS (Difco; 10 μg/ml) plus MCC (1 μM) were added to the cultured T cells for 1 d. Dead cells were removed by Lympholite-M (Cedarlane), and the CD4+ Treg cells were prestained by DyLight650-labeled anti-TCRβ (H57) Fab, conjugated with MCC-pulsed LPS-stimulated B cells for 5-10 min, fixed with 2% PFA, and imaged by confocal microscopy (Leica SP5) with ProLong gold antifade reagent with DAPI (Molecular Probes).
Kong K.F., Fu G., Zhang Y., Yokosuka T., Casas J., Canonigo-Balancio A.J., Becart S., Kim G., Yates JR I.I.I., Kronenberg M., Saito T., Gascoigne N.R, & Altman A. (2014). Protein Kinase C-η Controls CTLA-4-Mediated Regulatory T Cell Function. Nature immunology, 15(5), 465-472.
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4

Analyzing Brain Tumors in Mice

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Mouse showing neurological signs of late stage brain tumours or deemed endpoint by the clinical veterinarian were sacrificed under general anesthesia by exsanguination by the way of cardiac puncture. The collected blood (500 to 700ul) was quickly prepared for flow-cytometry using Lympholite-M (Cedarlane) accordingly to the manufacturer protocol. Brain tumors and spinal cord are quickly dissected and kept on ice until dissociation. Brain and spinal cords were imaged under a fluorescence stereoscope to measure extent of metastasis, the whole dorsal and ventral aspects of the CNS were examined. Upon stereoscopic examination the primary tumor samples as well as the spinal cords were quickly dissociated in Enzyme-Free cell dissociation solution (Thermo-Fisher) accordingly to the manufacturer recommendations. Dissociated cells were resuspended in PBS-1%BSA for flow cytometry stainings.
Garzia L., Kijima N., Morrissy A.S., De Antonellis P., Guerreiro-Stucklin A., Holgado B.L., Wu X., Wang X., Parsons M., Zayne K., Manno A., Kuzan-Fischer C., Nor C., Donovan L.K., Liu J., Qin L., Garancher A., Liu K.W., Mansouri S., Luu B., Thompson Y.Y., Ramaswamy V., Peacock J., Farooq H., Skowron P., Shih D.J., Li A., Ensan S., Robbins C.S., Cybulsky M., Mitra S., Ma Y., Moore R., Mungall A., Cho Y.J., Weiss W.A., Chan J.A., Hawkins C.E., Massimino M., Jabado N., Zapotocky M., Sumerauer D., Bouffet E., Dirks P., Tabori U., Sorensen P.H., Brastianos P.K., Aldape K., Jones S.J., Marra M.A., Woodgett J.R., Wechsler-Reya R.J., Fults D.W, & Taylor M.D. (2018). A Hematogenous Route for Medulloblastoma Leptomeningeal Metastases. Cell, 172(5), 1050-1062.e14.
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5

Inducing Arthritis in Chimeric Mice

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We irradiated 7-8-week-old C57BL/6 mice with divided two doses of 950 Rads (475 Rad each), using an irradiator with a 137Cs source and infused with a mixture of BM to generate 50:50 chimeras. Irradiated recipient mice were reconstituted, via retro-orbital injection, with 1 x 107 bone marrow cells collected from CD45.1 Dcstamp-/- and CD45.2 Tg(hTNF) mice. We isolated blood leukocytes by gradient density using Lympholite M (Cedarlane, Burlington, CA) and stained them with specific antibodies against CD45.2 and CD45.1 (Biolegend, San Diego, CA.) to verify 50:50 bone marrow cells reconstitution. 6-weeks after bone marrow reconstitution, we injected the mice at days 0 and 3 with 150 μl of serum collected from 8-week-old K/BxN mice. Twelve days after serum transfer, we euthanized the animals, and their hind paws were harvested for histology analysis or injected with an additional 150 µl of K/BxN serum on day 26 and sacrificed on day 30 for μCT analysis.
Garcia-Hernandez M.D., Rangel-Moreno J., Garcia-Castaneda M., Kenney H.M., Paine A., Thullen M., Anandarajah A.P., Schwarz E.M., Dirksen R.T, & Ritchlin C.T. (2022). Dendritic cell-specific transmembrane protein is required for synovitis and bone resorption in inflammatory arthritis. Frontiers in Immunology, 13, 1026574.
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