Macs cd34 microbead kit ultra pure

OP9 cells (a gift from the Mikkola Laboratory, UCLA) were cultured in alpha minimum essential medium (aMEM) with 2 mM L-glutamine, 1% pen-strep, 20% Hyclone (Thermo Fisher Scientific) fetal bovine serum (FBS). For OP9/leukocyte co-cultures, media was supplemented with 5 ng/mL thrombopoietin (TPO), 50 ng/mL SCF, 10 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3L), 5 ng/mL interleukin-6 (IL-6), and 5 ng/mL IL-3 (Peprotech). 32D cells (CRL-11346 American Type Culture Collection [ATCC]) were cultured according to ATCC recommendations with 10 ng/mL IL-3. Lenti-X HEK293 cells (632180 Clonetech) were cultured in DMEM 10% FBS. G1E-ER4 cells (a gift from the Ganz Laboratory, UCLA) were cultured with tamoxifen according to Rylski et al. (2003) (link). BV173, KCL22, and K562 cells (a gift from the Colicelli Laboratory, UCLA), Tf1a (ATCC CRL-2451) and HEL 92.1.7 (ATCC TIB-180) were all cultured according to ATCC recommendations.
Human PBMC and CD34⁺ cord blood cells were purchased from the UCLA/Core Facility Research (CFAR) Virology Core Laboratory. Mononuclear cells were isolated using a Ficoll gradient. Monocytes (PBMC fraction adhering to a TC plate) and lymphocytes (non-adhered portion) were used for RNA. For cord blood, CD34⁺ (positive selected) and CD34⁻ (negative selected) blood cells were isolated using human Miltenyi MACS CD34 MicroBead Kit Ultra Pure and used for RNA.

For in vivo studies, 6 to 8 week-old NOD scid gamma (NSG) mice were purchased from the Jackson laboratory (Bar Harbor, ME USA). The experimental protocol was approved by Stanford University’s Administrative Panel on Lab Animal Care. Sample sizes were not chosen to ensure adequate power to detect a pre-specified effect size. Four days after electroporation/transduction or directly after sorting, 500,000 cells (or 100,000-500,000 cells for the GFP^high group) were administered by tail-vein injection into the mice after sub-lethal irradiation (200 cGy) using an insulin Syringe with a 27 gauge x 1/2" needle. Mice were randomly assigned to each experimental group and evaluated in a blinded fashion. For secondary transplants, human cells from the RNP+AAV group were pooled and CD34⁺ cells were selected using a CD34 bead enrichment kit (MACS CD34 MicroBead Kit UltraPure, human, Miltenyi Biotec), and finally cells were injected into the femurs of female secondary recipients (3 mice total). Because GFP^high mice had low engraftment, they were not CD34⁺-selected, but total mononuclear cells were filtered, pooled, and finally injected into the femur of two secondary recipients.