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Blood, Vol. 113, Issue 17, 3953-3960, April 23, 2009
Human and murine amniotic fluid c-Kit+Lin– cells display hematopoietic activity
Blood Ditadi et al.
113: 3953
Supplemental materials for: Ditadi et al
Flow cytometry
The following mAbs were used for the phenotypic analysis of murine cells: CD117 PE- or APC-, Lin (described in Materials and Methods section) PE- of FITC-, CD34 FITC-, CD44 FITC-, Sca-1 FITC- and PE-, Thy1.2 FITC- and PE-, H2Kb FITC-, Prominin-1 APC- and Ly5.2 biotin-conjugated (revealed with a Avidin FITC-conjugated) Abs. All Abs were purchased from BD Biosciences, with the exception of anti-prominin (Miltenyi Biotech, Bergisch Gladbach, Germany). The phenotype of human AF cells was analyzed with the following mAbs: CD117 APC-, Lin (described above) PE-, CD34 FITC-, CD44 FITC-, CD90 PE- (all from BD), CD133 PE- (Miltenyi Biotec) and HLA-ABC FITC- or HLA-DR PE- (Dako, Trappes, France) conjugated antibodies. Myeloid differentiation in co-cultures of mKL and hKL cells was analyzed with Viaprobe and Gr1 APC-conjugated Ab for murine cells or CD33 APC- and CD13 PE-conjugated Abs for human cells. B cell differentiation cultures were analyzed by flow cytometry using a combination of IgM PE-, CD19 APC-conjugated Abs and Viaprobe. As with myeloid differentiation, successive side-scatter/forward-scatter (SSC/FSC) and 7AAD- gates were set in order to exclude stromal and non-viable cells from the analysis. After in vitro differentiation in T-cell conditions, a fraction of the culture was analyzed by flow cytometry using four different combinations of Abs: (i) CD25 PE- and CD44 APC-conjugated Abs (BD) (for murine cells only), (ii) CD5 FITC- and CD7 PE-conjugated Abs (BD) (for human cells only), (iii) CD8 PE- and CD4 APC-conjugated Abs (BD), (iv) TCRγδ PE- and CD3 APC-conjugated Abs (BD) and (iv) CD3 PE- and TCRαβ APC-conjugated Abs (BD), all in combination with Viaprobe. Successive SSC/FSC and 7AAD-GFP gates were set in order to exclude maternal contamination and OP9-hDelta1 stromal and non-viable cells from the analysis. The following mAbs were used for post-transplantation follow-up: B220 APC-, IgM PE-, CD4 PE- or APC- , CD3 APC- or PE-, CD8 PE, Gr1 APC-, Mac1 PE-, CD44 APC-, CD25 PE-, NK1.1 APC, Sca1 PE-, cKit APC-, Ter119 PE-, Ly5.2 peridinin-chlorophyll-protein complex-(PerCP) and Ly5.1 PerCP- conjugated Abs. All these Abs and their isotypes were purchased from BD.
Files in this Data Supplement:
- Table S1. Primers and product sizes for gene expression analysis (PDF, 22.4 KB) -
The sets of primers used in Fig. 5 are detailed. All used the 28S rRNA gene as an endogenous control. Reverse transcription reactions were carried out using primer A. The first amplification reaction used primers A and B, giving rise to the initial products which were then amplified separately using primers A and C to generate secondary products. The respective sizes of the initial and secondary products are indicated.
- Table S2. Phenotype of murine and human KL cells (PDF, 16.5 KB) -
Murine KL cells from the AF, the Am and FL were stained with antibodies specific for CD34, prominin-1, CD90.2, H2Kb, Ly5.2, CD44, and Sca-1. Human AF- and CB-KL cells were stained with specific mAbs for CD34, CD133, CD90, HLA-ABC, CD45, CD44, and HLA-DR. For each marker indicated in the left column, the mean percentage of positive cells and standard deviation were determined.
- Figure S1. Dot plot showing GFP and 7AAD controls (unstained cells issued from a C57BL/6J wt mouse) used as a basis to draw the gate GFP+/7AAD− in Fig. 1A (JPG, 4.37 KB)
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