Abstract
This short communication describes a three-color flow cytometric analysis procedure for terminal deoxynucleotidyl transferase (TdT). The method combines CD45-gating principles and a new permeabilization method based on a commercially prepared lysing reagent. While traditional permeabilizing fixatives are often cumbersome to use, distort cell light scatter, and fail to retain the fluorochrome peridin chlorophyll alpha protein (perCP), this simple method retains light scatter and does not affect perCP, making it ideal for three-color analysis. The procedure simply involves staining whole blood or bone marrow with CD45 perCP and an appropriate phycoerythrin (PE) conjugated monoclonal antibody (MoAb) and lysing with FACS lysing solution (Becton Dickinson, San Jose, CA). After washing the cells, fluorescein isothiocyanate (FITC) conjugated anti-TdT is added. The cells are washed, fixed with formaldehyde, and acquired on a flow cytometer compensated for three-color analysis. Display of CD45 perCP vs. right angle light scatter (RALS) allows identification of blasts. Dual color expression of anti-TdT FITC and a PE conjugated MoAb identifies TdT expression on blast populations defined by specific lineage associated antigens. This method is not only useful for TdT analysis but may also prove to be a valuable tool for looking at expression of other cytoplasmic antigens in combination with surface antigens on CD45-defined blast populations.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 228-230 |
| Number of pages | 3 |
| Journal | Cytometry |
| Volume | 18 |
| Issue number | 4 |
| DOIs | |
| State | Published - 1994 |
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Keywords
- CD45 perCP
- FACS lysing solution
- Leukemia immunophenotyping
- Permeabilization
ASJC Scopus subject areas
- Hematology
- Cell Biology
- Pathology and Forensic Medicine
- Biophysics
- Endocrinology
Cite this
Detection of terminal deoxynucleotidyl transferase by flow cytometry : A three color method. / Horvatinovich, J. M.; Sparks, S. D.; Borowitz, Michael J.
In: Cytometry, Vol. 18, No. 4, 1994, p. 228-230.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Detection of terminal deoxynucleotidyl transferase by flow cytometry
T2 - A three color method
AU - Horvatinovich, J. M.
AU - Sparks, S. D.
AU - Borowitz, Michael J
PY - 1994
Y1 - 1994
N2 - This short communication describes a three-color flow cytometric analysis procedure for terminal deoxynucleotidyl transferase (TdT). The method combines CD45-gating principles and a new permeabilization method based on a commercially prepared lysing reagent. While traditional permeabilizing fixatives are often cumbersome to use, distort cell light scatter, and fail to retain the fluorochrome peridin chlorophyll alpha protein (perCP), this simple method retains light scatter and does not affect perCP, making it ideal for three-color analysis. The procedure simply involves staining whole blood or bone marrow with CD45 perCP and an appropriate phycoerythrin (PE) conjugated monoclonal antibody (MoAb) and lysing with FACS lysing solution (Becton Dickinson, San Jose, CA). After washing the cells, fluorescein isothiocyanate (FITC) conjugated anti-TdT is added. The cells are washed, fixed with formaldehyde, and acquired on a flow cytometer compensated for three-color analysis. Display of CD45 perCP vs. right angle light scatter (RALS) allows identification of blasts. Dual color expression of anti-TdT FITC and a PE conjugated MoAb identifies TdT expression on blast populations defined by specific lineage associated antigens. This method is not only useful for TdT analysis but may also prove to be a valuable tool for looking at expression of other cytoplasmic antigens in combination with surface antigens on CD45-defined blast populations.
AB - This short communication describes a three-color flow cytometric analysis procedure for terminal deoxynucleotidyl transferase (TdT). The method combines CD45-gating principles and a new permeabilization method based on a commercially prepared lysing reagent. While traditional permeabilizing fixatives are often cumbersome to use, distort cell light scatter, and fail to retain the fluorochrome peridin chlorophyll alpha protein (perCP), this simple method retains light scatter and does not affect perCP, making it ideal for three-color analysis. The procedure simply involves staining whole blood or bone marrow with CD45 perCP and an appropriate phycoerythrin (PE) conjugated monoclonal antibody (MoAb) and lysing with FACS lysing solution (Becton Dickinson, San Jose, CA). After washing the cells, fluorescein isothiocyanate (FITC) conjugated anti-TdT is added. The cells are washed, fixed with formaldehyde, and acquired on a flow cytometer compensated for three-color analysis. Display of CD45 perCP vs. right angle light scatter (RALS) allows identification of blasts. Dual color expression of anti-TdT FITC and a PE conjugated MoAb identifies TdT expression on blast populations defined by specific lineage associated antigens. This method is not only useful for TdT analysis but may also prove to be a valuable tool for looking at expression of other cytoplasmic antigens in combination with surface antigens on CD45-defined blast populations.
KW - CD45 perCP
KW - FACS lysing solution
KW - Leukemia immunophenotyping
KW - Permeabilization
UR - http://www.scopus.com/inward/record.url?scp=0028631175&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028631175&partnerID=8YFLogxK
U2 - 10.1002/cyto.990180407
DO - 10.1002/cyto.990180407
M3 - Article
C2 - 7895530
AN - SCOPUS:0028631175
VL - 18
SP - 228
EP - 230
JO - Cytometry
JF - Cytometry
SN - 0196-4763
IS - 4
ER -