Dynabeads™ CD34 Positive Isolation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The CD34+ fraction was isolated by magnetic bead method as per manufacturer's instructions	Isolated CD34+ cells were expanded for 3 days in StemPro-34 SFM (Invitrogen, Grand Island, NY, USA) serum free media with SCF, IL-6, TPO and Flt3L (25 ng/ml each, Peprotech, Princeton, NJ, USA).

Protocol tips
The CD34+ fraction was isolated by magnetic bead method as per manufacturer's instructions
Downstream tips
Isolated CD34+ cells were expanded for 3 days in StemPro-34 SFM (Invitrogen, Grand Island, NY, USA) serum free media with SCF, IL-6, TPO and Flt3L (25 ng/ml each, Peprotech, Princeton, NJ, USA).

Publication protocol

The CB was collected from AFMC, Pune and mononuclear cell fraction was separated by Ficoll-Hypaque (density 1.077 g/ml from Himedia, India) density gradient centrifugation. The CD34+ fraction was isolated by magnetic bead method as per manufacturer's instructions (Dynabeads CD34 positive isolation kit, Invitrogen, Grand Island, NY, USA). Isolated CD34+ cells were expanded for 3 days in StemPro-34 SFM (Invitrogen, Grand Island, NY, USA) serum free media with SCF, IL-6, TPO and Flt3L (25 ng/ml each, Peprotech, Princeton, NJ, USA). 106 expanded cells were nucleofected in Opti-MEM media (Gibco, Grand Island, USA) using 1 μg of Epi5 Episomal iPSC Reprogramming Kit plasmids (Invitrogen, Grand Island, NY, USA) by U-008 program in Amaxa Nucleofector (Lonza Walkersville, MD, USA). The cells were resuspended in StemPro-34 SFM containing same growth factors and set on geltrex (Invitrogen, Grand Island, NY, USA) coated plates for 24 h. After this, cells were maintained in DMEM/F-12 media containing 1% N2 supplement, 2% B27 supplement, 1% non essential amino acid (NEAA) (All from Invitrogen, Grand Island, NY, USA) and 100 ng/ml bFGF (Peprotech, Princeton, NJ, USA) for 8 days by demi-depopulation. On day 9, the spent media was replaced by Essential 8 (Invitrogen, Grand Island, NY, USA) with media change daily until colonies emerged. From here onwards, the cells were maintained in Essential 8 media. The colonies were scored for calculating transfection efficiency and manually picked after day 25 onto freshly coated plates and were passaged every 4–5 days.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Dynabeads™ CD34 Positive Isolation Kit below.

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