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Lymphocyte isolation from various tissues (mouse, NHP and human)

eagle-i ID

http://eagle-i.itmat.upenn.edu/i/0000014b-7f73-7c58-d034-1ff680000000

Resource Type

  1. Material processing service

Properties

  1. Fee for service
    Yes
  2. Resource Description
    "Isolation of lymphocytes from the different compartments is critical to the study and understanding of the immune response to viral vectors and transgene products. <strong>Blood collection and isolation of peripheral blood mononuclear cells (PBMCs).</strong> PBMCs are isolated from whole blood collected in EDTA-containing Vacutainer tubes after percoll density-gradient centrifugation. Cells are collected from the interphase and washed with PBS. PBMCs are incubated with of ACK lysing buffer to lyse RBCs. Cells are washed again and resuspended in complete RPMI medium (Mediatech) containing 10% FBS and 2 mM glutamine. <strong>Isolation of lymphocytes from liver.</strong> Tissue sections of the liver weighting 20 grams are placed in RPMI 1640 medium, diced into 5 mm peaces and washed with PBS. Tissue fragments are diced is smaller fragments of 1mm3 in RPMI+ Collagenase type 1a, crushed on stainless steel mesh and incubated at 37oC. Digested tissue is passed thru a 40 um nylon mesh, washed with PBS and resuspended in RPMI. Lymphocytes are isolated after after 30-70% percoll density-gradient. Cell are collected from the interphase, washed with PBS and resuspended in complete RPMI medium. <strong>Isolation of lymphocytes from lymphoid nodes and spleen.</strong> Tissue sections of the spleen and lymph nodes are crushed on 40 um stainless steel meshes. Filtered cells are washed with PBS, incubated with ACK lysing buffer, washed again and resuspended in complete RPMI medium. <strong>Isolation of lymphocytes from Bone Marrow.</strong> Bone marrow samples from crashed Femure and humerus are colleted in PBS containing heparin. Lymphocytes are isolated after percoll density-gradient centrifugation. Cells are collected from the interphase, washed and resuspended in complete RPMI medium. <strong>Isolation of lymphocytes from gut.</strong> Biopsy samples are washed with EDTA and incubated with HBSS in continuous agitation. Supernatant is removed and intraepithelial lymphocytes (IELs) are isolated. The settled biopsies are incubated with a collagenase solution on a rocker. Collagenase digestion is used to isolate Lamina Propria Lymphocytes (LPLs). IELs and LPLs are enriched using Percoll density gradient centrifugation. <img src="http://www.med.upenn.edu/gtp/user_images/clip_image014.gif" alt="Figure 7 from core website"> Figure 7 : Isolation and immunophenotyping of lymphocytes from blood, gut and liver and immunophenotyping using 2 different panels of memory markers: CD28-CD95 and CD62L-CD45RA. CM: central memory, EM: effector memory, TD: terminally differentiated."
  3. Service Fee URL
    https://somapps.med.upenn.edu/pbr/portal/gimm/fees.php
  4. Topic
    Homo sapiens
  5. Topic
    Mus musculus
  6. Service Provided by
    Immunology Core (Penn)
  7. Website(s)
    http://www.med.upenn.edu/gtp/immunology_tcells.shtml
  8. Related Technique
    Cell separation
 
RDFRDF
 
Provenance Metadata About This Resource Record
  1. workflow state
    Published
  2. contributor
    fcoldren
  3. created
    2015-02-12T15:23:04.908-05:00
  4. creator
    fcoldren
  5. modified
    2015-02-12T15:43:34.838-05:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016