DA-endothelial cells (DA-ECs) were isolated by fluorescence-activated cell sorting (FACS).23 (link) DA tissues from ≈30 rat fetuses on gestational day 21 were subjected to FACS to obtain one sample. We obtained 6 samples for quantitative reverse transcription polymerase chain reaction. Dispersed cells by collagenase enzyme mixture were subjected to FACS analysis. Fluorescein isothiocyanate (FITC)–conjugated anti-CD31 antibody (Abcam, Cambridge, United Kingdom) and APC/Cy7–conjugated anti-CD45 antibody (BioLegend, San Diego, MA) were used as cell surface markers for ECs and hematopoietic derivation cells, respectively. CD31+/CD45− and CD31−/CD45− were defined as DA-ECs and DA-non-ECs. This analysis was conducted using BD FACS AriaII (Becton Dickinson, San Jose, CA).
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Isolation of DA-Endothelial Cells by FACS
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Anti antibody
Cells
Collagenase
Endothelial cells
Enzyme
Fetuses tissues
Fluorescein
Gestational
Hematopoietic
Isothiocyanate
Reverse transcription polymerase chain reaction
Corresponding Organization :
Other organizations : Yokohama City University, Tokyo Medical University, Augusta University, Oita University, Institute for Molecular Science, Aichi Medical University, Kanagawa Children's Medical Center
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Variable analysis
independent variables
- Isolation of DA-endothelial cells (DA-ECs) by fluorescence-activated cell sorting (FACS)
- Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of the 6 samples obtained
- DA tissues from approximately 30 rat fetuses on gestational day 21
- Dispersed cells by collagenase enzyme mixture
- FITC-conjugated anti-CD31 antibody and APC/Cy7-conjugated anti-CD45 antibody as cell surface markers for ECs and hematopoietic derivation cells, respectively
- CD31+/CD45- defined as DA-ECs and CD31-/CD45- defined as DA-non-ECs
- BD FACS AriaII used for analysis
- No positive or negative controls were explicitly mentioned in the input protocol.
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