Single-cell suspensions of the spleen, mLN, and a pool of scLN (Lnn. mandibularis, Lnn. cervicales superficiales, Lnn. axillares et cubiti, Lnn. inguinales superficiales, and Lnn. subiliaci) were prepared using 70-µm cell strainers (BD). mAbs to CD4 (RM4-5, GK1.5), CD8 (53–6.7), CD25 (PC61, 7D4), CD62L (MEL-14), pSTAT5 [Clone 47 recognizing phosphorylated Y694 of Stat5a (Stat5(pY694))] and Pacific Blue- and PE-conjugated streptavidin were obtained from eBioscience or BD. Before FACS, for some experiments, CD4+ or CD8+ cells were enriched from single-cell suspensions using biotinylated mAbs directed against CD4 or CD8, respectively, streptavidin-conjugated microbeads and the AutoMACS magnetic cell separation system (Miltenyi Biotec). Samples were analyzed on an LSRII or sorted using a FACS Aria II or FACS Aria III (BD). Data were analyzed using FlowJo software (Tree Star, Inc.).
Automacs magnetic cell separation system
Manufactured by Miltenyi Biotec
Sourced in Germany
The AutoMACS magnetic cell separation system is a laboratory instrument designed for the automated separation of cells. It utilizes magnetic beads coated with antibodies to target and isolate specific cell types from complex biological samples. The system offers a standardized and efficient method for cell separation, facilitating the purification of desired cell populations for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd8a ly 2 microbeads, by Miltenyi Biotec (2 mentions)
70 m cell strainer, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Erythrocyte lysis buffer, by Qiagen (1 mentions)
Lsrfortessa system, by BD (1 mentions)
Mel 14, by Thermo Fisher Scientific (1 mentions)
70 μm cell strainer, by BD (1 mentions)
4 protocols using automacs magnetic cell separation system
1
Isolation and Analysis of Immune Cells
2
Isolation of CD8+ Splenocytes
3
Isolation of CD8+ Splenocytes
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Pools of splenocytes from individual groups were incubated with anti-mouse-CD8+ antibodies and separated as recommended by the manufacturer’s instructions using the autoMACS magnetic cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, mouse spleen cells were incubated with CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). After incubation, cells were washed with 2 mL of buffer (0.25% BSA, 0.5 M EDTA), and centrifuged at 300 × g for 10 min. Pellets were resuspended in buffer and samples were loaded in the autoMACS.
4
Isolation and Analysis of Immune Cells
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Single-cell suspensions of spleen, mesenteric lymph nodes or a pool of subcutaneous lymph nodes (Lnn. mandibularis, Lnn. cervicales superficiales, Lnn. axillares et cubiti, Lnn. inguinales superficiales, and Lnn. subiliaci) were prepared using 70 μm cell strainers (BD Biosciences). Bone marrow cells were harvested from femurs and tibias by flushing the cavities of intact bones with flow cytometry buffer (Hank's buffered salt solution, supplemented with 5% fetal calf serum and 10 mM HEPES), followed by filtration through 70 μm cell strainers. Single-cell suspensions from the spleen and bone marrow were subjected to red blood cell lysis (erythrocyte lysis buffer, Qiagen). Monoclonal antibodies to CD4 (RM4-5, GK1.5), CD8 (53–6.7), CD19 (1D3), CD25 (PC61, 7D4), CD69 (H1.2F3), CXCR4 (2B11), CCR7 (4B12), CCR9 (CW-1.2), CD62L (MEL-14), and CD44 (IM 7) and PE- and PE-Cy7-conjugated streptavidin were obtained from ThermoFisher (eBioscience) or BD Biosciences. Before flow cytometry, for some experiments, CD4+ or CD25+ cells were enriched from single-cell suspensions using biotinylated antibodies directed against CD4 or CD25, respectively, streptavidin-conjugated microbeads, and the AutoMACS magnetic cell separation system (Miltenyi Biotec). Samples were analyzed on an LSRII or LSR Fortessa system (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).
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