After Ficoll separation, CD34+ cells from cord blood were isolated with EasySep human CD34 selection kit (STEMCELL Technologies, Vancouver, Canada, https://www.stemcell.com ) and cultured in StemSpan medium (STEMCELL Technologies), supplemented with cytokines.
Easysep human cd34 selection kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep human CD34 selection kit is a laboratory product designed to isolate and enrich human CD34+ cells from various sample types, such as peripheral blood, bone marrow, or cord blood. The kit utilizes the EasySep positive selection technology to magnetically label the target cells, which can then be separated using an EasySep magnet.
Automatically generated - may contain errors
Lab products found in correlation
Stem cell factor (scf), by Miltenyi Biotec (2 mentions)
Methocult sf h4236, by STEMCELL (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Low density lipoprotein, by STEMCELL (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by Miltenyi Biotec (2 mentions)
Stemspan medium, by STEMCELL (1 mentions)
Transit reagent, by Mirus Bio (1 mentions)
Histopaque, by Merck Group (1 mentions)
6 protocols using easysep human cd34 selection kit
1
CD34+ Cell Isolation and Culture
2
Isolation and Colony Counting of CD34+ Cells
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The EasySep human CD34+ selection kit (Cat#:18056, StemCell Tech, Canada) was used to isolate CD34+ cells from frozen BM cells. The purity of CD34+ cells was greater than 90%. The CD34+ cells were then counted and seeded in complete methylcellulose (Methocult H04034; Stem Cell Technologies) at a concentration of 500 cells/mL and plated in 35-mm grid plates, 1 mL/plate, in triplicate per mouse according to the manufacturer’s instructions. Colonies were counted 2 weeks later in a blinded fashion using a QImaging Micropublisher 3.3 CCD digital camera and QCapture software version 3.0 (QImaging, Surrey, BC).
3
Generation and Culture of MRGPRX2+ PBCMCs
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Human PBCMCs were generated as described previously (18 (link)). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of healthy donors or rocuronium-hypersensitive patients using Histopaque. Next, CD34+ progenitors were isolated from PBMCs (EasySep Human CD34 Selection Kit, Stemcell Technologies) and cultured, for 4-5 weeks, in serum-free methylcellulose-based medium (MethoCult SF H4236, Stemcell Technologies) supplemented with penicillin (100 units/mL, Life Technologies), streptomycin (100 μg/mL, Life Technologies), low-density lipoprotein (LDL, 10 μg/mL, Stemcell Technologies), 2-mercaptoethanol (55 μmol/L, Life Technologies), stem cell factor (SCF, 100 ng/mL, Miltenyi Biotec), interleukin-3 (IL-3, 100 ng/mL, PeproTech) and interleukin-6 (IL-6, 50 ng/mL, Miltenyi Biotec). PBCMCs harbor a MRGPRX2+ and a MRGPRX2- subpopulation (Supplementary Figure 1
4
Retroviral Transduction of CD34+ Cells
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Retoviral vector expressing hTERT (pBabe-puro-hTERT) and control empty vector (pBabe-puro) were obtained from Dr. Robert Weinberg (Addgene). To produce retrovirus, 1.5ug of each construct was transfected respectively into phoenix-gp cells on 6 well plates along with pSV-ampho-env (1.0ug) and pEQ-PAM3(-E) (0.5ug) using TransIT reagent (Mirus). Supernatant was collected at 36 and 48 hours. CD34+ cells were selected from week 6-8 old AE pre-leukemia cultures using EasySep human CD34 selection kit (Stemcell Technology). Retronectin (Takara Shuzo) coated plates were preloaded with the virus supernatant by spinoculation at 2000 g for 2hr at 22°C, and then loaded with CD34+ AE cells suspended in fresh viral supernatant. 8 hours later, transduced cells were harvested, expanded for 2 days, and then selected with 0.4ug/ml puromycin for 1 week.
5
Isolation and Culture of PBCMC Subpopulations
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Human PBCMCs were cultured as previously described 5, 8 . Briefly, peripheral blood mononuclear cells were isolated from 50 mL of fresh peripheral blood from healthy volunteers, and CD34 + progenitor cells were enriched using the EasySep™ Human CD34 Selection Kit (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. Isolated peripheral blood CD34 + progenitor cells were cultured in a serum-free methylcellulose-based medium (MethoCult™ SF H4236; STEMCELL Technologies) supplemented with penicillin (100 units/mL; Life Technologies, Waltham, MA, USA), streptomycin (100 mg/mL; Life Technologies), low-density lipoprotein (10 mg/mL; STEMCELL Technologies), 2-mercaptoethanol (55 mmol/L; Life Technologies), stem cell factor (100 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), interleukin-3 (100 ng/mL; PeproTech, Rocky Hill, NJ, USA), and interleukin-6 (50 ng/mL; Miltenyi Biotec) for 4-5 weeks.
PBCMCs harbour a MRGPRX2 + and a MRGPRX2 -subpopulation, relevant to our further experiments (Suppl. Figure 1).
PBCMCs harbour a MRGPRX2 + and a MRGPRX2 -subpopulation, relevant to our further experiments (Suppl. Figure 1).
6
Isolation of CD34+ Progenitor Cells
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CD34 + progenitor cells were isolated from peripheral blood as described in 29 (link) . Briefly, CD34 + progenitor cells were purified via a multi-step process. First, mononuclear cells were isolated by density gradient separation (Histopaque, Merck, St. Louis, Missouri, USA). Then, CD34 + progenitor cells were magnetically separated using the EasySep Human CD34 Selection Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer's instructions.
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