Multiparameter flow cytometric quantification of apoptosis-related protein expression. A van Stijn, A Kok, MA van der Pol, N Feller, GMJM Roemen, AH Westra, ...
Leukemia (2003) 17, 787–788 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu
APPENDIX: METHOD IN FOCUS Multiparameter flow cytometric quantification of apoptosis-related protein expression A van Stijn, A Kok, MA van der Pol, N Feller, GMJM Roemen, AH Westra, GJ Ossenkoppele and GJ Schuurhuis Department of Haemotology, VU University Medical Centre, Amsterdam, The Netherlands
Leukemia (2003) 17, 787–788. doi:10.1038/sj.leu.2402886
Assay characteristics The method is developed for accurate analysis of intracellular proteins in combination with extracellular antigens. The method enables detection and quantification of apoptosis-related proteins in a bulk of cells, but more importantly in very small cell populations as has been demonstrated in this paper for Bcl2, Bcl-xL, Mcl-1 and Bax expression in de novo AML cells and in minimal residual disease (MRD) cells in BM samples of AML patients at several intervals after start of therapy.
Protocol
BM sample preparation
saponin for 15 min at RT and washed. After permeabilisation, an incubation for 30 min at 41C with either 50 ml FITC-conjugated mouse anti-human Bcl-2 (1:20) or polyclonal nonconjugated rabbit anti-human Bax (1:20), Bcl-xL (1:20) or Mcl-1 (1:20) is performed. For Bcl-2, cells are washed and analysed immediately. Cells that are incubated with the nonconjugated antibodies against Bax, Bcl-xL, and Mcl-1 are washed, and incubated for another 30 min at 41C with FITC-conjugated antirabbit IgG (1:20). Following the second step incubation, cells are washed and analysed immediately on the flow cytometer.
Quantification In order to quantitate the levels of fluorescence in LAP+cells, relative apoptosis-related protein expression is calculated using the following formula: Protein ratio ½LAPþ cells ¼
Mononuclear cells from BM samples of de novo patients were isolated by ficoll gradient centrifugation, whereafter residual erythrocytes were lysed with ammonium chloride for 10 min at 41C and washed. In follow-up AML BM samples erythrocytes were lysed with ammonium chloride for 10 min at 41C and washed. All nucleated cells were used for the pursuing staining procedure.
Extracellular staining Cells (0.1–1 106) are labelled for 15 min at room temperature with PE-, PerCP-, and APC-conjugated MoAbs (5 ml/MoAb) defining a leukemia-associated phenotype (LAP), consisting of aberrant antigen combinations that are not or in very low frequences present in normal BM.1–9 In all cases, the FL1 channel is reserved for subsequent labelling of the intracellular proteins using FITC-conjugated antibodies.
MFIprotein ½LAPþ cells=MFIIsotype control ½LAPþ cells
Time requirement Total time: 2.5–5 h. The time indicated includes preparation of cells (0–2 h), bench work (2 h), measurement on flow cytometer (10–30 min) and analysis (10–30 min).
Materials
Hardware FACS Calibur flow cytometer equipped with a Red Diode Laser (Becton Dickinson, Mountain View, CA, USA).
Software Intracellular staining After the surface labelling for the LAP, cells are washed with PBS+0.1% BSA and fixed in 100 ml 1% paraformaldehyde during 5 min at RT. Hereafter, cells are permeabilised with 100 ml 0.1% Correspondence: Dr GJ Schuurhuis, Department of Haemotology, VU University Medical Centre, BR240, PO Box 7057, Amsterdam 1007 MB, The Netherlands; Fax: +31 20 444 2601 Received 6 September 2002; accepted 16 December 2002
Cellquest and Paint-A-gate are both from Becton and Dickinson.
Reagents and solutions Saponin was from Sigma-Aldrich Chemie (Steinheim, Germany).
Antibodies: FITC-conjugated mouse anti-human Bcl-2 (clone 124) and FITC-conjugated anti-rabbit were from Dako
Quantifying apoptosis-related protein expression A van Stijn et al
788 Diagnostics BV (Glostrup, Denmark). The rabbit polyclonal antibodies Bax (P-19), Bcl-xL (S-18) and Mcl-1 (S-19) were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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