< OMIPS Directory
OMIP-059

Identification of Mouse Hematopoietic Stem and Progenitor Cells with Simultaneous Detection of CD45.1/2 and Controllable Green Fluorescent Protein Expression by a Single Staining Panel

Marcus Eich, Andreas Trumpp, Steffen Schmitt Cytometry PART A, Volume 95, Issue 10, 1049-1052 (2019)

PURPOSE: The panel was developed and optimized to identify mouse bone marrow hematopoietic stem cells (HSCs) and five multipotent progenitors (MPPs) along with controllable green fluorescent protein (GFP) expression as well as CD45.1 and CD45.2. HSCs and MPPs can be identified by analyzing the cell surface proteins Sca1, cKit, CD150, CD48, CD34, and CD135. The common myeloid progenitor (CMP), the granulocyte macrophage progenitor (GMP), the megakaryocyte erythrocyte progenitor (MEP), and the common lymphoid progenitor (CLP) are defined by specific expression patterns of Sca1 and cKit plus CD16/32 and CD34, CD135, and CD127. Additionally, the membrane integrity is determined with a life/dead discriminator to differentiate living from dead cells. The staining is performed in freshly isolated living cells without fixation obtained from transgenic mice (SCL‐tTA H2B‐GFP or ISRE‐EGFP) expressing the GFP and the genetic alleles CD45.1 or CD45.2. The analysis of the status of these markers is included in the reported 16 parameter staining panel.

CELL TYPE: Hematopoietic stem and precursor cells

MACHINE: BD FACSAria Fusion

Marker Target Clone Fluorochrome Purpose
Dead cells Mouse Live/Dead Fix Near IR (775) Viability
CD4 Mouse Gk1.5 PE-Cy7 Lineage
CD8a Mouse 53-6.7 PE-Cy7 Lineage
CD11b Mouse M1/70 PE-Cy7 Lineage
CD45R (B220) Mouse RA3-6B2 PE-Cy7 Lineage
Ly-6G/Ly-6C Mouse RB6-8C5 PE-Cy7 Lineage
TER-119 Mouse TER119 PE-Cy7 Lineage
Ly-6A/Ly-6E Mouse D7 Brilliant Violet 421 LSK, LS-K
CD117 Mouse 2B8 PE LSK, LS-K
CD150 Mouse TC15-12F12.2 Brilliant Violet 785 HSC, MPP
CD48 Mouse HM48-1 BUV395 HSC, MPP
CD34 Mouse RAM34 Alexa Fluor 700 LT-HSC, ST-HSC, GMP, CMP, MEP
Flt-3 Mouse A2F10 APC MPP3, MPP4,CLP
cd16/32 Mouse 2.4G2 Brilliant Violet 480 CMP,GMP,MEP
CD127 Mouse A7R34 Brilliant Violet 650 CLP
CD45.1 Mouse A20 PE-CF594 internal reporter
CD45.2 Mouse 104 BUV737 internal reporter