Isolation and labeling of cells with the barcoding library was performed as previously described (18 (link)). Briefly, after isofluorane anesthesia and cervical dislocation, bone marrow cells were isolated from femur, tibia and iliac bones of 2 donor mice per 4 recipient mice by flushing, and c-Kit+ cells were enriched by MACS with anti-CD117 magnetic beads (Miltenyi). Cells were stained for antibodies against CD117, CD135, CD150, Sca-1 and MPP4 were sorted using a FACSAria (BD Biosciences) IIIu as CD117+/Sca–1+/CD150–Cd135+ (see Figure 2 ) and transduced with the barcode library in StemSpanMedium SFEM (STEMCELL Technologies) with 50 ng/ml mSCF (STEMCELL Technologies) through 1.5 h of centrifugation at 300 g and 4.5 h incubation at 37°C to obtain ∼10% barcoded cells. After incubation, the cells were transplanted by tail vein injection into recipient mice that were 6 Gy sub-lethally irradiated using an X-ray generator 3 h prior to the transplantation.
Anti cd117 magnetic beads
Manufactured by Miltenyi Biotec
Anti-CD117 magnetic beads are laboratory reagents used for the isolation and separation of cells expressing the CD117 (c-Kit) antigen. These beads are coated with antibodies specific to the CD117 surface marker, allowing for the targeted capture and isolation of CD117-positive cells from complex biological samples.
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Lab products found in correlation
Facsaria, by BD (1 mentions)
Ter119, by BioLegend (1 mentions)
Cd3ε clone 145 2c11, by BioLegend (1 mentions)
Cd45r b220 clone ra3 6b2, by BioLegend (1 mentions)
Cd11c clone n418, by Thermo Fisher Scientific (1 mentions)
Cd3 clone 17a2, by BioLegend (1 mentions)
Ter 119 clone ter119, by BD (1 mentions)
Nk1.1 clone pk136, by BioLegend (1 mentions)
Cd11b clone m1 70, by BioLegend (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Iodixanol, by Axis-Shield (1 mentions)
3 protocols using anti cd117 magnetic beads
1
Isolation and Barcoding of Hematopoietic Stem Cells
2
Isolation and Characterization of Murine Hematopoietic Stem Cells
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BM was harvested from femurs, tibias, and ilia, and cells were enriched using anti-CD117 magnetic beads (Miltenyi). The c-kit+ fraction was stained with antibodies against CD117 (c-kit APC, clone 2B8, Biolegend, dilution 1/100), Sca-1 (APC-Cy7, clone D7, Biolegend, 1/100), CD135 (Flt3 PE-Cy5, clone A2F10, Life technologies, 1/50), CD150 (Slam Pecy7, clone TC15-12F12.2, Biolegend, 1/100), CD48 (Pacific Blue, HM48-1, Biolegend, 1/100), CD16/32 (PercPCy5.5, clone M1/702.4G2, eBioscienceBD Bioscience, 1/100), CD34 (Alexa 700, 1/100), and a lineage cocktail (PE, CD3ε clone 145-2C11; Ly-6G/Ly-6C clone RB6-8C5; CD11b clone M1/70; CD45R/B220 clone RA3-6B2; TER-119, Biolegend, 1/200). The c-kit− fraction was stained with antibodies against CD11b (PercPCy5.5, clone M1/70, eBioscience, 1/100), Ly6C (APC, HK1.4, Thermofisher, 1/200), Ly6G (BV510, RUO, Biolegend, 1/100), Siglec F (PE CF594, RUO, BD 1/100), F4/80 (alexa 700, clone BM8, Ozyme, 1/100), in addition to a lineage cocktail in PeCy7 (B220 clone RA3-6B2 Biolegend, CD3, clone 17A2 Biolegend, CD11c clone N418, eBioscience, NK1.1 clone PK136 Biolegend, Ter119 clone TER-119 BD Biosciences) and DAPI (1/1000) as live/dead marker. All cells were analyzed on a Ze5 (Bio-Rad) plate-reader cytometers. Data analysis was performed using FlowJo v10.2 software (TreeStar) and Prism v9.
3
Isolation of Liver Progenitor Cells
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Cells were isolated as described in a previous study.4 (link) Briefly, mice were anesthetized and perfused with Hank’s buffer containing 0.2 mg/mL of collagenase IV (Sigma-Aldrich). Hepatocytes were removed by low-speed centrifugation at 50 × g for 3 minutes. Liver NPCs were resuspended in 4 mL of 17.6% iodixanol (Axis-Shield, Dundee, Scotland) and 4 mL of 10% iodixanol. The cell suspension then was centrifuged at 1400 × g for 20 minutes in a swing-out rotor without brake. Cells were recovered from the interface between the 10% and 17.6% iodixanol solutions, and resuspended and incubated with anti-CD117 magnetic beads (Miltenyi Biotec) for 15 minutes in the dark in the refrigerator. By magnetic separation with MS Columns (Bergisch Gladbach, Germany), c-kit+ cells were collected by pushing the plunger into the MS column. The eluted fraction can be enriched and incubated with anti-CD146 magnetic beads, upon which magnetic separation obtains the c-kit- cells.
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