Basophil isolation kit

Ficoll density gradient centrifugation was used to separate peripheral blood mononuclear cells (PBMCs) from the buffy coats of healthy donors (Centre Trinité, L’Établissement Français du Sang, Paris; EFS-INSERM ethical committee permission 18/EFS/033). The negative selection method was used to obtain the purified basophils from the PBMCs by using the Basophil Isolation Kit (Basophil Cell Isolation Kit II, human (Catalogue: 130-092-662) Miltenyi Biotec, Paris, France) according to the manufacturer’s protocol.

Human basophils were isolated from the buffy bags of healthy donors (Centre Trinité, L’Établissement Français du Sang, Paris; EFS-INSERM, 18/EFS/041) as previously described (28 (link)) by using Basophil Isolation Kit (Miltenyi Biotec, Paris, France). Basophils were then cultured in X-Vivo medium, with 100 ng/0.5 M cells/ml of IL-3, or with 10 nM all-trans RA for 6 hr with or without prior treatment with 1 µM each of retinoic acid receptors (RAR) antagonists CD2665 (RARβ/γ antagonist; Tocris, Cat. 3800) and BMS614 (RARα antagonist; Sigma, Cat. SML-1084) for 1 hr or with RAR antagonists for 1h followed by IL-3 for 6h or with RAR antagonists alone for 1h. Untreated basophils (Baso alone) were used as control.
Total RNA from the different experimental conditions was isolated using the RNeasy minikit (Qiagen, Hilden, Germany). cDNAs were synthesized using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Courtaboeuf, France), and quantitative PCR was performed with LightCycler 480 (Roche Diagnostics) and QuantiTect SYBR Green Kit (Qiagen) using the primers as described in Table 3. Relative RNA levels were calculated with human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) as an internal control.