Multicolor immunofluorescence and flow cytometry utilizing cascade blue to purify murine hematopoietic stem cells from fetal liver and bone marrow.
BACKGROUND
Here we demonstrate the utility of cascade blue (CB), to purify hematopoietic stem cells by flow cytometry. Multicolor immunofluorescence and the sensitivity (signal-to-noise) of the fluorochromes are essential for the identification and isolation of rare stem cell populations.


METHODS
We isolated hematopoietic stem cells utilizing a 407 nm laser line to excite CB and propidium iodide (PI) in combination with FITC, PE, and Red670 which were excited at 488 nm.


RESULTS
CB is maximally excited using a 407 nm laser line, when compared to UV or 413 nm excitation. The increase in sensitivity of CB at 407 nm can be contributed to higher absorption of CB and a reduction of autofluorescence at this excitation wavelength (Ropp et al.: Cytometry 21: 309-317, 1995).


CONCLUSIONS
Despite the fact that the CB antibody conjugate has a tendency to adhere specifically to a B cell subpopulation in bone marrow, we nevertheless could purify stem cells by using CB for the detection and elimination of lineage positive cells. Isolated stem cells from mouse fetal liver (Lin-CD34(+)Sca-1(+)c-Kit(high)) and adult bone marrow (Lin-CD34(-/low)Sca-1(+)c-Kit(+)) were transplanted into lethally irradiated mice, and the sorted stem cells had the ability to efficiently repopulate all mature hematopoietic lineages in recipient mice.
Donahue
C J
CJ
Department of Immunology, Genentech, Inc., South San Francisco, California, USA.
Fennie
C
C
Villacorta
R
R
La
H
H
Lasky
L A
LA
Ohneda
O
O
D000818:Animals; D000918:Antibody Specificity; D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells / Q000737:chemistry; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005333:Fetus / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000737:chemistry*; D006801:Humans; D008099:Liver / Q000196:embryology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009942:Organometallic Compounds / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D011247:Pregnancy; D014184:Transplantation, Homologous
D018952:Antigens, CD34; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; C066850:Cascade Blue