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ROR1 is a receptor tyrosine kinase family member studied for its roles in development and cancer. Here we describe a protocol for analysis of ROR1 surface expression in acute lymphoblastic leukemia immortalized cell lines by flow cytometry.

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ROR1 Flow Cytometry

Cell Biology > Cell-based analysis > Flow cytometry
Authors: Vincent T. Bicocca
Vincent T. BicoccaAffiliation: Hematology and Medical Oncology, Oregon Health and Science University, Portland, USA
Bio-protocol author page: a841
 and Jeffrey W. Tyner
Jeffrey W. TynerAffiliation: Cell, Developmental and Cancer Biology Department, Oregon Health and Science University, Portland, USA
For correspondence: tynerj@ohsu.edu
Bio-protocol author page: a842
Vol 3, Iss 18, 9/20/2013, 1976 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.905

[Abstract] ROR1 is a receptor tyrosine kinase family member studied for its roles in development and cancer. Here we describe a protocol for analysis of ROR1 surface expression in acute lymphoblastic leukemia immortalized cell lines by flow cytometry.

Materials and Reagents

  1. Cells (e.g. RCH-ACV cells, Kasumi-2 cells, REH cells, MHH-CALL-2 cells)
  2. FBS
  3. Antibody specific for ROR1 (R&D Systems, catalog number: AF2000)
  4. Goat IgG (R&D Systems, catalog number: AB-108-C)
  5. Donkey Anti-goat IgG-Phycoerythrin (R&D Systems, catalog number: F0107)

Equipment

  1. Centrifuge
  2. FACSAria (BD Biosciences)

Procedure

  1. Actively cultured RCH-ACV, Kasumi-2, REH, and MHH-CALL-2 cells were pelleted and washed once in PBS and then resuspended in PBS wash buffer containing 2% FBS (1 million cells in 50 μl of buffer).
  2. 1 x 106 cells were immunostained at room temperature for 30 min with 1 μg of antibody specific for ROR1 or Goat IgG (do not need to rotate the reaction).
  3. Cells were washed 3 times with 500 μl PBS wash buffer.
  4. Cells were stained with Donkey Anti-goat IgG-Phycoerythrin (10 μl Donkey Anti-goat IgG-Phycoerythrin is diluted into 90 μl PBS wash buffer). Incubate at room temperature in the dark for 15 min.
  5. Samples are washed 1x with 500 μl PBS wash buffer and then resuspended in 200 μl PBS wash buffer for analysis.
  6. Samples were analyzed on a BD FACSAria.

Acknowledgments

This protocol was adapted from Bicocca et al. (2012).

References

  1. Bicocca, V. T., Chang, B. H., Masouleh, B. K., Muschen, M., Loriaux, M. M., Druker, B. J. and Tyner, J. W. (2012). Crosstalk between ROR1 and the Pre-B cell receptor promotes survival of t(1;19) acute lymphoblastic leukemia. Cancer Cell 22(5): 656-667.


How to cite this protocol: Bicocca, V. T. and Tyner, J. W. (2013). ROR1 Flow Cytometry. Bio-protocol 3(18): e905. DOI: 10.21769/BioProtoc.905; Full Text



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