Prostate cancer cells (1 × 106 cells) were injected into male CB.17. SCID mice (4–6 weeks of age: Charles River, Wilmington, MA) by intracardiac or intratibial injection. Bone marrow cells were flushed from femurs and tibias 24 h later. Single cell preparations were incubated first with a Lineage Cell Depletion Kit magnetic labeling system with biotinylated anti-Lineage (CD5, CD45R (B220), CD11b, Gr-1 (Ly-6G/C), and Ter-119) antibody cocktail (130-092-613, Miltenyi Biotec) and anti-Biotin MicroBeads (130-090-485, Miltenyi Biotec), and then enriched for murine Lineage negative population using an AutoMACS machine (Miltenyi Biotec). The enriched cells were incubated with a FITC- anti-HLA-ABC antibody, PE-anti-CD133 antibody, and APC-anti-CD44 antibody for another 20 min at 4°C. Thereafter, the CD133+/CD44+ and CD133−/CD44− fractions were sorted with a FACSAria II Cell Sorter by gating on HLA-ABC positive cells. All experimental procedures were approved by the University of Michigan Committee for the Use and Care of Animals.
Automacs machine
Manufactured by Miltenyi Biotec
The AutoMACS Pro Separator is a fully automated magnetic cell separation system designed for high-purity cell isolation. The system utilizes magnetic microbeads that bind to target cells, which are then separated magnetically. The AutoMACS Pro Separator automates the cell separation process, providing consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Anti biotin microbead, by Miltenyi Biotec (2 mentions)
Mouse cd3ε microbead kit, by Miltenyi Biotec (2 mentions)
Cb17 scid mice, by Charles River Laboratories (1 mentions)
Histopaque gradient, by Merck Group (1 mentions)
Facsaria 2, by BD (1 mentions)
Ter119, by Thermo Fisher Scientific (1 mentions)
Cd16 32, by Thermo Fisher Scientific (1 mentions)
Sca 1, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd117 microbeads, by Miltenyi Biotec (1 mentions)
Cd150, by Thermo Fisher Scientific (1 mentions)
Mac 1, by Thermo Fisher Scientific (1 mentions)
Streptavidin magnetic beads, by Miltenyi Biotec (1 mentions)
Anti cd45r b220 biotin, by Miltenyi Biotec (1 mentions)
7 protocols using automacs machine
1
Isolation of Prostate Cancer Stem Cells
2
Isolating Bone Marrow and Peripheral Blood Cells
3
Murine Hematopoietic Stem Cell Isolation
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Bone marrow cells were harvested by crushing two tibias, two femurs, two pelvises, and one spine from each mouse. Bone marrow cells were enriched for immature cells using anti–mouse CD117 MicroBeads and an autoMACS machine (both Miltenyi Biotec) per manufacturer’s instructions. c-Kit–enriched populations were stained with antibodies against lineage markers (B220, CD3, Gr-1, Mac-1, and Ter119), c-Kit, Sca-1, CD150, CD16/32, and CD34 (eBioscience) as previously described (McGowan et al., 2011 (link)). Stained samples were either analyzed or sorted using a FACSAria II (BD). For all experiments in which c-Kithi or c-Kitlo HSCs were purified, they were double-sorted to ensure >95% purity. To calculate the frequency of hematopoietic precursors, two femurs and two tibias were flushed into 1x PBS containing 2.5% fetal calf serum (Hyclone). Bone marrow aspirations were performed on mice under isoflurane anesthesia per IACUC-approved protocol. Aspirates were treated with ACK lysis buffer and stained in PBS/2.5% fetal calf serum with antibodies against lineage markers, c-Kit, Sca-1, CD150, CD16/32, Flk2, CD34, CD48, and CD41 for analysis on hematopoietic stem and progenitor cells. For myeloid progenitors, bone marrow cells were stained with antibodies against lineage markers, c-Kit, Sca-1, CD150, CD16/32, CD41, CD105, and CD71, as described by Pronk et al. (2007) (link).
4
CD45.1+ TCR75 Cell Enrichment
5
Isolation of CD4+ T cells from H. pylori-infected mice
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CD4+ T cells were isolated from the spleens of H. pylori-infected IL-21−/− mice and H. pylori-infected wild-type littermates between 2 and 3 months postinfection. Spleens were harvested, and after red blood cell lysis, the cells were magnetically labeled with CD4 microbeads (clone L3T4; Miltenyi Biotec). CD4+ cells were positively selected using the positive selection program in sensitive mode on the autoMACS machine (Miltenyi Biotec) according to the manufacturer’s protocol. The resulting population was 92 to 96% CD4+ by flow cytometry analysis.
6
Generating Chimeric Mice via Irradiation and Cell Transfer
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To make chimeras, recipient mice were irradiated with x rays (two doses of 4.5 Gy given 4 h apart) and then injected i.v. with at least 2 × 106 BM cells that had been depleted of T cells using magnetic beads (Mouse CD3ε Microbead Kit; Miltenyi) and the “Depl05” program on an autoMACS machine (Miltenyi). To purify T reg cell cells for adoptive transfer, pooled spleen and lymph node cells from Foxp3GFP mice were incubated with anti-CD45R(B220)-biotin (catalog no. 130–101-998; Miltenyi) and anti-CD8α-biotin (catalog no. 130–118-074; Miltenyi), followed by incubation with anti-biotin MicroBeads (catalog no. 130–090-485; Miltenyi) to allow removal of B cells and CD8+ T cells using the “Deplete” program on an autoMACS machine, before 1 × 105–2 × 105 FACS-sorted viable CD4+ GFP+ cells were injected i.v. per recipient.
7
Generating TCR-retrogenic Mice
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DNA encoding the 6218 TCRα and TCRβ genes separated by the “cleavable” P2A peptide were synthesised (Genscript Biotech, Piscataway, NJ). Cys-encoding codons were introduced into the CDR3α (Ser110α replaced by Cys110α) or CDR3β (Gly109β replaced by Cys109β) sequence using PCR mutagenesis. DNA constructs were cloned into the pMSCV-IRES-GFP II (pMIG II) vector, which encodes GFP under the control of an internal ribosomal entry site35 (link). Rag1–/– BM cells were retrovirally transduced in vitro, as described previously35 (link),63 (link). B6 BM was depleted of T cells using a mouse CD3ε MicroBead Kit (Miltenyi, Bergisch Gladbach, Germany, Cat. no. 130-094-973) and an autoMACS machine (Miltenyi). Rag1–/– BM cells and T cell-depleted B6 BM cells were mixed 1:1 before i.v. injection of > 2 × 106 cells per Rag1–/– or B6 recipient, which had been irradiated with x-rays (two doses of 5 Gy given 4 h apart) earlier in the day. Tcra–/– BM donors and recipients, or Zap70mrd/mrt BM donors and recipients, were used to make TCR-retrogenic mice with the same protocol, except that T cell-depleted B6 BM cells were not administered in these cases.
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