BM cells were incubated with APC-conjugated anti–c-Kit antibody, and c-Kit+ cells were enriched using anti-APC magnetic beads and LS columns (Miltenyi Biotec). The positively selected cells were then stained for HSC markers using a lineage cocktail as described in the previous section and Alexa Fluor 700–conjugated anti-CD34, Brilliant Violet 605–conjugated anti-CD150 (BioLegend), PE-Cy7–conjugated anti-CD48 (BioLegend), APC-conjugated anti–c-Kit, and PE-conjugated anti–Sca-1 (BioLegend) antibodies and streptavidin-APC-Cy7. After staining, cells were sorted on a cell sorter (FACSAria III; BD).
Anti apc magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany, Canada
Anti-APC magnetic beads are a laboratory product designed for cell isolation and purification. The beads are coated with antibodies specific to the APC (Allophycocyanin) fluorescent dye, allowing for the isolation of APC-labeled cells from a complex sample. The magnetic properties of the beads enable efficient separation and enrichment of the target cells using a magnetic field.
Automatically generated - may contain errors
Lab products found in correlation
Ls column, by Miltenyi Biotec (3 mentions)
Dnase, by Thermo Fisher Scientific (3 mentions)
Facsaria 2, by BD (2 mentions)
Usinganti cd138 apc clone 281 2, by BioLegend (2 mentions)
Apc conjugated anti c kit, by BioLegend (1 mentions)
Pe conjugated anti sca1, by BioLegend (1 mentions)
Apc conjugated anti c kit antibody, by Miltenyi Biotec (1 mentions)
Facsaria 3, by BD (1 mentions)
Facscelesta instrument, by BD (1 mentions)
Histopaque 1083, by Merck Group (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Facsaria sorter, by BD (1 mentions)
Magnisort negative selection beads, by Thermo Fisher Scientific (1 mentions)
Facsdiva 8, by BD (1 mentions)
Gentlemacs c tube, by Miltenyi Biotec (1 mentions)
Sca 1, by Thermo Fisher Scientific (1 mentions)
Ter119, by Thermo Fisher Scientific (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Anti cd20 microbeads, by Miltenyi Biotec (1 mentions)
21 protocols using anti apc magnetic beads
1
Isolation and Characterization of Murine Hematopoietic Stem Cells
2
Isolation and Analysis of 2W1S-Specific CD4+ T Cells
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Spleens and mesenteric lymph nodes (mLNs) were made into single-cell suspensions by homogenizing organs over 100 µm nylon mesh filter in cold sorter buffer (1× phosphate buffered saline, 2% newborn calf serum, 0.1% sodium azide). Single-cell preparations were resuspended in 200 µL with FcBlock. Cells were enriched as previously described [52 (link)] by staining with 10 µM 2W1S:1-Ab MHC-II tetramer, conjugated to allophycocyanin (APC), incubated at room temperature in the dark for 1 h, washed, stained with anti-APC magnetic beads (Miltenyi, Bergisch Gladback, Germany), and passed over an LS column on a quadroMACS magnet. Eluted cells were stained with the following anti-mouse antibodies: CD4 (PE), CD44 (PerCP-Cy5.5), CD8 (BV510), CD3 (fluorescein isothiocyanate [FITC]), CD11b, CD11c, CD19, and F4/80 (eFlour 450). Cells were collected on a BD FACSCelesta instrument (BD, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
3
Isolation and Staining of LLO-specific CD4 T Cells
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Single cell suspensions were isolated from collagenase digested or mechanically dispersed MLNs and spleens. Cells from LP and IEL compartments were isolated as previously described.19 (link) 5–10×106 cells were stained with LLO190-201 I-Ab streptavidin (SA)-phycoerythrin (PE) or LLO190-201 I-Ab SA-allophycocyanin (APC) MHC class II tetramers in IMDM medium supplemented with 10% FBS and 50nM Dasatinib for 1 h at 37°C. LLO I-Ab tetramers were kindly provided by Dr. Marc Jenkins (University of Minnesota, Minneapolis, MN). In some experiments tetramer enrichment was used for quantification of LLO-specific CD4 T cells. In these experiments, cells were further incubated with anti-PE or anti-APC magnetic beads (Miltenyi Biotec, Auburn, CA) for positive selection through a magnetic column. Ova257-264 H-2Kb SA-APC MHC class I tetramers were made from MHC class I monomers provided by the NIH Tetramer Core Facility. MHC I tetramer staining was performed at room temperature for 1 h. Surface antigens were then identified with antibodies for 30 min at 4°C.
4
Isolation of Mouse Bone Marrow CD31+ Cells
5
Adoptive transfer of alveolar macrophages
6
Isolation of Mouse Hematopoietic Stem Cells
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CD150+CD34−Kit+Sca1+Lineage− HSCs were isolated from mouse BM by fluorescence-activated cell sorting (FACS)16 (link),17 (link). Briefly, WBMCs were stained with APC-cKit antibody and cKit-positive cells enriched using anti-APC magnetic beads and LS columns (Miltenyi Biotec). The cKit-enriched cells were then stained with a biotin lineage antibody cocktail (biotin-CD4, -CD8, -CD45R/B220, -TER119, -Gr1, and -CD127), before being stained with anti-CD34, anti-cKit, anti-Sca1, anti-CD150, and streptavidin-APC/eFluor780 for 90 min (see Supplementary Table 1 for antibody details). Cells were purified using a FACS AriaII (BD) and BD FACS Diva 8 software by direct sorting into wells containing HSC media using PI as a live/dead stain (see Supplementary Fig. 3A for representative FACS gating).
7
Murine Dendritic Cell Culture
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Bone marrow from the dissected femurs of adult male CBA/Ca mice was depleted of erythrocytes and cultured at 7.5 × 105 cells/mL in 90 mm diameter tissue culture plates in R10 medium (RPMI 1640 supplemented with 10% fetal calf serum [FCS], 2 mM L‐glutamine, 1% sodium pyruvate and 50 μM 2‐mercaptoethanol) further supplemented with ~5 ng/mL murine GM‐CSF harvested from the supernatant of an X6310 cell line stably transfected with the murine Gmcsf gene. Nonadherent cells were removed on days 3 and 6 of culture when the medium was replaced and cells were harvested on day 7. DCs were purified using anti‐CD11c‐APC monoclonal antibodies (mAb) followed by anti‐APC magnetic beads, according to the manufacturer's instructions (Miltenyi Biotec, Bisley, Surrey, UK).
8
Adoptive Transfer of Splenic B Cells
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Splenic CD138hi B cells were enriched on day 5 p.i. using
anti-CD138-APC (clone 281–2, BioLegend) and anti-APC magnetic beads
(Miltenyi). CD19+CD138hiIgDneg plasmablasts and
CD19+CD138loIgDneg activated B cells were
sort purified. 1–2 × 106 cells were transferred i.v. to
mice on day 7 p.i. Mice that did not receive cells were given 200 μL of
PBS i.v. on day 7. Sera and spleens were harvested from recipients on day 21
p.i. (day 14 post-transfer). Parasitemia was monitored every 2–3 days
p.i.
anti-CD138-APC (clone 281–2, BioLegend) and anti-APC magnetic beads
(Miltenyi). CD19+CD138hiIgDneg plasmablasts and
CD19+CD138loIgDneg activated B cells were
sort purified. 1–2 × 106 cells were transferred i.v. to
mice on day 7 p.i. Mice that did not receive cells were given 200 μL of
PBS i.v. on day 7. Sera and spleens were harvested from recipients on day 21
p.i. (day 14 post-transfer). Parasitemia was monitored every 2–3 days
p.i.
9
Isolation of Skeletal Muscle Precursor Cells
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Satellite cells were isolated by methods described previously (Bareja et al., 2014 (link)). In brief, dissected muscle was digested in gentleMACS C tubes (Miltenyi Biotec). subjected to dissociation using a GentleMACS dissociator (Miltenyi Biotec). After digestion, samples were filtered, spun, re-suspended, and transferred to a 5-mL fluorescence-activated cell sorting (FACS) tube for staining. Cells were stained with the following monoclonal antibodies—CD11b (1:100), CD31 (1:100), CD45 (1:100), Ter119, and Sca1 (all conjugated to APC, eBioscience), CD34 (1:50), phycoerythrin (BD Biosciences), and ITGA7 (FITC, R&D Scientific). Following a 45-min-long incubation on ice, the cell suspension was spun down and incubated with anti-APC magnetic beads (1:10, Miltenyi Biotec) for 15 min on ice. All cells bound to APC-conjugated antibodies were separated from the original suspension using the manual MACS cell separation protocol (Miltenyi Biotec). Mouse skeletal muscle precursors were isolated by FACS using CD34 and ITGA7 as positive selection markers while CD31, CD45, CD11b, Sca1, and Ter119 were used as negative selection markers (for further details, see Supplemental Experimental Procedures ).
10
CD4+ T cell Isolation and Purification
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CD4+ T cells were enriched from PBMC of healthy donors or active TB patients by negative selection method using CD4+ T Cell Isolation Kit II from Miltenyi (130-096-533). Then, CD4+ T cells were stained with anti-human CD161-APC (BD, 550968), followed by anti-APC magnetic beads (Miltenyi Biotech, 130-090-855) for secondary positive purification. Cells were loaded onto columns and the passing cells were collected as CD4+CD161- T cells, CD4+CD161+ T cells were released from columns using release buffer (Miltenyi Biotech). There was no evidence for significant activation ofCD4+CD161+ T cells after purification (data not shown). B cells were positivity isolated from PBMC using anti-CD20 microbeads from Miltenyi Biotech according to the manufacturer’s instructions. Cell purity was consistently ≥96% as demonstrated in Suppplementary Figure 1A
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