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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.1/1.4


TITLE: IMMUNOPHENOTYPING


INTRODUCTION

Human cell lines obtainable from the DSMZ are analyzed routinely for expression of differentiation and activation marker proteins. The suspension cell lines are screened for expression of cell surface markers with a set of monoclonal antibodies (mAb) classified according to the CD system. Cell lineage and, to some extent, also the differentiation status of the cells can be detected by these means. Other human cell lines (besides leukemia-lymphoma cells) are checked for the presence of intermediary filaments. These filaments are specific for cells from different tissues. Thus, detection of intermediary filaments and cell surface markers helps to prevent misidentifications of cell lines, and it facilitates the characterization of the cells.

PROCEDURE

Immunophenotyping includes a two-step staining procedure:

  1. Antigen-specific murine mAb are added to the cells.
  2. Binding of the mAb is assessed by an immunofluorescence technique using FITC-conjugated anti-mouse Ig antisera.
  3. Distribution of antigens is analyzed by flow cytometry and/or light microscopy.
  4. Explanation of symbols: -, negative; (+), weakly positive or not consistently positive; +, positive. Intensity of antigen expression may vary between passages and may be influenced by cell culture conditions.
  5. The following mAb are used in our panel:
    CD1 (NA1/34), CD2 (RFT-11), CD3 (Leu-4), CD4 (S3.5 or RFT-4), CD5 (RFT-1), CD6 (RFT-12), CD7 (RFT-2), CD8 (RFT-8), CD10 (RFAL-3), CD13 (MY7 or MCS-2), CD14 (VIM-13 or FMC-17 or MY4), CD15 (Leu-M1 or VIM-D5), CD19 (RFB-9), CD20 (B-1), CD21 (RFB-6), CD22 (RFB-4), CD25 (Tac), CD30 (HRS-1 or HRS-4), CD33 (MY9), CD34 (HPCA-1), CD37 (RFB-7), CD38 (OKT-10), CD41 (SZ22), CD42b (SZ2), CD45 (CBL-124), CD68 (EBM.11), CD71 (L01.1), CD138 (ID4), HLA-DR (RFDR-2 or I3), TCR / (WT31), TCR / (11F2), goat anti-human IgM, IgG, Ig , and Ig (sm = surface membrane or cy = intracytoplasmatic), anti-glycophorin A (VIE-G4), anti-human neurofilament protein (2F11), anti-human glial fibrillary acid protein (RPN.1106), anti-vimentin (V9), anti-human desmin (D33), anti-human endothelial cell (CD31; JC/70A), anti-human cytokeratin (MNF116).
    Non-reactive IgG and IgM antibodies are applied as controls.


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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Copyright CABRI, 1998

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