1. Apoptotic Regulation in Primitive Hematopoietic Precursors
by Rowayda Peters, Serge Leyvraz, and Lucien Perey
Blood
Volume 92(6):
September 15, 1998
©1998 by American Society of Hematology

2. FACS analysis of MPB mononuclear cells.
FACS analysis of MPB mononuclear cells. (A) Forward scatter (size) and side scatter (density) of MPB MNCs showing region R1 in which CD34+ cells are located. (B) Scattogram of CD34-tri-color (Fl 3) versus side scatter (SSC) showing only CD34+ cells in R2 that were also gated in R1. (C and D) CD34-tri-color (Fl 3) and CD38-PE (Fl 2) expression from R2 showing the gating of CD34+ cells based on CD38 antigen density level (R3, R4, and R5). Region R3 used to define CD34+/CD38−/low cells matching the biological control (see Materials and Methods). Region R3 include CD34+ cells with PE-CD38 fluorescence more by 20% to 25% than the maximum PE fluorescence of irrelevant isotype-control (log 101). Region R4 defines CD34+/CD382+ and region R5 defines CD34+/CD383+ cells. Regions R3, R4, and R5 were stored and constantly used for all samples to analyze the third antigen and for cell sorting experiments. (C) The gating of only CD34 strong positive cells are shown, where greater than 98% of CD38−/low cells are located in region R3. (D) The gating of CD34 weak positive cells are shown, in which region R3 lack the CD38− cells.
Rowayda Peters et al. Blood 1998;92:
©1998 by American Society of Hematology

3. Bimodal distribution of bcl-2 protein expression (bcl-2low and bcl-2high) in primitive hematopoietic precursors and their progeny.
Bimodal distribution of bcl-2 protein expression (bcl-2low and bcl-2high) in primitive hematopoietic precursors and their progeny. Mononuclear cells (MNCs) were stained with membrane antigens (CD34-tri-color and CD38-PE), then permeabilized with Ortho Permeafix and stained for anti-bcl-2 FITC. Cells were analyzed according to CD38 intensity level, CD38−/low (R3), CD382+ (R4), and CD383+ (R5) by using only the strong CD34 positive cells (Fig 1C). These selected gates were identically used for analyzing all samples. MNCs were also permeabilized and stained with mIgG to define the threshold between bcl-2-positive and bcl-2-negative cells. Similarly, the granulocytes from whole, fresh MPB samples were also analyzed. The bcl-2 gates (bcl-2low and bcl-2high) were decided by the histograms distribution. (A) The bimodal distribution of bcl-2 in MPB, CB, and BM. (B) The comparison in bcl-2 fluorescent intensity between the mature granulocytes and the hematopoietic precursors (CD34s+/CD38−/low) seen in Fig 2A.
Rowayda Peters et al. Blood 1998;92:
©1998 by American Society of Hematology

4. Rowayda Peters et al. Blood 1998;92:2041-2052
©1998 by American Society of Hematology

5. Rowayda Peters et al. Blood 1998;92:2041-2052
©1998 by American Society of Hematology

6. Rowayda Peters et al. Blood 1998;92:2041-2052
©1998 by American Society of Hematology

7. Histograms of bcl-x-FITC and bcl-xL-FITC staining after the immunomagnetic selection of CD34+ and CD34+/CD38− cells from MPB. The left-hand histogram represents the negative control (mIgG), the middle empty histogram is the CD34+ cells expressing bcl-x and ...
Histograms of bcl-x-FITC and bcl-xL-FITC staining after the immunomagnetic selection of CD34+ and CD34+/CD38− cells from MPB. The left-hand histogram represents the negative control (mIgG), the middle empty histogram is the CD34+ cells expressing bcl-x and the filled-up histogram is the CD34+/CD38−hematopoietic precursors expressing high intensity bcl-xLprotein. Cells stained for bcl-x and bcl-xL were first stained with anti-CD34-PE antibody then permeabilized with Ortho Permeafix. MNCs stained with mIgG were also permeabilized.
Rowayda Peters et al. Blood 1998;92:
©1998 by American Society of Hematology

8. Flow cytometric analysis of pro-apoptotic proteins expressed by KG-1A cell line.
Flow cytometric analysis of pro-apoptotic proteins expressed by KG-1A cell line. Cells were induced for apoptosis after overnight incubation in medium deprived of serum. For flow cytometric analysis of bak, bad, and bax expression in KG-1A cells, cells were permeabilized with Ortho Permeafix and stained with goat anti-bak, mouse anti-bad, and rabbit anti-bax antibodies followed by staining with FITC-conjugated antigoat, antimouse, and antirabbit IgG antibodies, respectively. For neutralization, the anti-bak antibody (goat polyclonal) was incubated overnight at 4°C with excess peptide antigen. Cells were stained with the peptide/antibody mixture followed by staining with FITC-conjugated antigoat IgG. (A) bax, bak, and bad expression before induction of apoptosis. Only bad protein was expressed by the majority of KG-1A cells. (B) Considerable increase in bax and bak expression following induction of apoptosis. (C) The effect of bak- blocking peptide (PEP) on bak expression in apoptotic cells. Bak expression reduced from 78% before to 11% after neutralization with bak peptide.
Rowayda Peters et al. Blood 1998;92:
©1998 by American Society of Hematology

9. ASP expression in CD34 positive cells obtained from MPB samples gated according to cell size into small, intermediate, and large.
ASP expression in CD34 positive cells obtained from MPB samples gated according to cell size into small, intermediate, and large. Region R6 identifies weak CD34 positive cells expressing the ASP-protein, and region R7 identifies strong CD34 positive cells expressing the ASP-protein. Three-color immunofluorescence staining was performed. After staining of membrane antigens with anti-CD34-tri-color and anti-CD38-PE antibodies, cells were permeabilized with Ortho Permeafix, stained with anti-ASP-FITC, and analyzed on the FACScan.
Rowayda Peters et al. Blood 1998;92:
©1998 by American Society of Hematology

10. Flow cytometric analysis of propidium iodide stained CD34+/CD38− cells obtained from MPB. (A) The immunomagnetic separation of CD34+/CD38− cells obtained from selected CD34+ cells.
Flow cytometric analysis of propidium iodide stained CD34+/CD38− cells obtained from MPB. (A) The immunomagnetic separation of CD34+/CD38− cells obtained from selected CD34+ cells. Greater than 90% of CD34+cells were CD38+. The MFI for CD38+ cells was 675. After negative selection, the MFI for CD38−cells reduced to 15. (B) Histogram showing PI-fluorescence of selected CD34+/CD38− cells stained with two-color immunofluorescence (CD34-FITC and PI) and gated on CD34+cells. M1 defines cells in G0/G1 phase, M2 defines cells in S phase, and M3 defines cells in G2+M phase.
Rowayda Peters et al. Blood 1998;92:
©1998 by American Society of Hematology

11. Changes in bcl-2 expression of CD34+/CD38−/low hematopoietic precursors during overnight incubation of MPB in serum-free RPMI. Cells were analyzed using three-color immunofluorescence of CD34-tri- color, CD38-PE, and bcl-2-FITC.
Changes in bcl-2 expression of CD34+/CD38−/low hematopoietic precursors during overnight incubation of MPB in serum-free RPMI. Cells were analyzed using three-color immunofluorescence of CD34-tri- color, CD38-PE, and bcl-2-FITC. After membrane antigen staining with anti-CD34 and anti-CD38 antibodies, cells were permeabilized with Ortho Permeafix and stained for anti-bcl-2. Bcl-2 expression was analyzed according to CD38 antigen intensity level (regions R3, R4, and R5 in Fig 1C). Data show one representative experiment of six. (A) Bcl-2 expression before incubation, representing a bimodal histogram of bcl-2low(MFI = 95) and bcl- 2high (MFI = 259). (B) Bcl-2 expression in the same cells after incubation showing a single histogram of bcl- 2high (MFI = 212).
Rowayda Peters et al. Blood 1998;92:
©1998 by American Society of Hematology