This procedure was essentially described in [31 (link)]. To obtain virions issued from CD45 expressing cells (CD45 is indeed mostly a T cell marker), SupT1 stably expressing Flag-IFITMs were challenged with replication-competent NL4-3 at an MOI of 3 to induce a rapid burst of infection and robust production of virion particles. Virions produced upon ongoing infection were harvested 6 days after infection and were incubated with magnetic beads coupled with an anti-CD45 antibody (Miltenyi) for 2 hours. Beads were then recuperated on a magnetic support following the manufacturer’s instructions. Bound and unbound material was analyzed by WB.
Anti cd45 antibody
Manufactured by Miltenyi Biotec
Sourced in United States
The Anti-CD45 antibody is a laboratory reagent used for the identification and isolation of cells expressing the CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated hematopoietic cells. This antibody can be used in various cell analysis and separation techniques.
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Lab products found in correlation
Facsaria 2, by BD (3 mentions)
Uea 1, by Vector Laboratories (2 mentions)
Epcam, by BioLegend (2 mentions)
Dnase 1, by Roche (2 mentions)
Liberase, by Roche (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Anti dclk1, by Abcam (1 mentions)
Anti aire, by Thermo Fisher Scientific (1 mentions)
Podoplanin, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Anti epcam antibody, by Miltenyi Biotec (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Magnetic column, by Miltenyi Biotec (1 mentions)
Collagenase b, by Roche (1 mentions)
Magnetic bead, by Miltenyi Biotec (1 mentions)
Diva software, by BD (1 mentions)
Rpmi medium, by Lonza (1 mentions)
Fc block, by Miltenyi Biotec (1 mentions)
8 protocols using anti cd45 antibody
1
Purification of HIV-1 Virions from CD45+ Cells
2
Isolation and Analysis of Thymic Epithelial Cells
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For the analysis of thymic epithelial cells (TECs), minced thymuses were digested with 0.5 unit/ml Liberase (Roche) in the presence of 0.02% DNase I (Roche). Single-cell suspensions were stained with antibodies specific for EpCAM (BioLegend), CD45 (eBioscience), Ly51 (Biolegend), Podoplanin (Biolegend), I-Ab (BioLegend), and for the reactivity with UEA-1 (Vector Laboratories). For the analysis of Aire and DCLK1, surface-stained cells were fixed in 5% formaldehyde neutral buffer solution (Nacarai Tesque), permeabilized in 1x permeabilization buffer (eBioscience), and stained with anti-Aire (eBioscience) or anti-DCLK1 (abcam) antibody. For the isolation of TECs, CD45− cells were enriched in magnetic bead conjugated anti-CD45 antibody (Miltenyi Biotec). Multicolor flow cytometry and cell sorting were performed on FACSVerse, FACSAria II, and LSRFortessa (BD Biosciences).
3
Enrichment of EpCAM-positive CTCs
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After the gradient test, the mononuclear cells were incubated with 100 μl of micromagnetic beads coated with anti-EpCAM antibody (Miltenyi-Biotec) and 100 μl of FcR-blocking reagent (Miltenyi-Biotec) for 30 minutes at 4°C and then incubated with 20 μl of magnetic beads coated with anti-CD45 antibody (Miltenyi-Biotec) for 15 minutes at 4°C. The mixture was passed through a magnet-filled column with an AutoMACSPro cell separator system (Miltenyi-Biotec) using the positive selection protocol (POSSELD program) to enrich for EpCAM-positive cells and the negative selection protocol (DEPLETE program) to enrich for CD45-depleted CTCs. The CD45-depleted (i.e., CD45-negative) fraction underwent an additional run through the magnet-filled column using the MACS DEPLETES program to prevent any residual contamination of CD45-positive cells.
4
Skeletal Muscle Macrophage Isolation
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Skeletal Muscles collected from hind limb were minced and digested enzymatically and mechanically in a single cell suspension with 0.2% of Collagenase B (Roche) in RPMI medium (Lonza) for 1 h and 30 min in a water bath at 37 °C under agitation. After filtration with cell strainers (70 and 40 µm, Grainer) and centrifugation at 272 × g for 10 min at 4 °C, the single cell suspension was resuspended in sterile PBS-0.5% BSA (Genespin)-2 mM EDTA (Sigma-Aldrich) and incubated with anti-CD45 antibody conjugated with magnetic beads (Miltenyi Biotech) for 30 min at 4 °C. Cells were then washed with PBS-0.5% BSA (Genespin)-2 mM EDTA (Sigma-Aldrich) and CD45+ cell isolation was performed by using magnetic columns (Miltenyi, Biotech) according to manufacturer instructions. After Fc blocking (Fc Buffer, Miltenyi Biotech), the fraction of CD45+ cells was incubated with Ly6C-PE antibody (eBioscence), to discriminate pro-inflammatory (Ly6C+) from anti-inflammatory macrophages (Ly6C−), and with CD64-APC antibody (BD Bioscience) to discriminate neutrophils from macrophages. Cell sorting experiments were then performed using a FACSAria II (BD Bioscience). Diva software (BD Pharmingen, San Diego, CA) was used for data acquisition and analysis.
5
Macrophage Phenotyping in Muscle Regeneration
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Macrophage phenotype was analyzed as previously described (13 (link)). Briefly, CD45+ cells were isolated from regenerating TA muscle using magnetic beads conjugated to anti-CD45 antibody (Miltenyi Biotec) and then incubated with Fc block (Miltenyi Biotec) for 30 minutes at 4°C. Finally, CD45+ cells were stained with antibody against Ly6C/G (17-5931-82, eBioscience) and against F4/80 (12-4801-82, eBioscience). Percentages of Ly6C/GhiF4/80lo and Ly6C/GnegF4/80hi cells were calculated among total F4/80+ cells after analysis by flow cytometry with a FACSCalibur instrument (BD Biosciences) and FlowJo version 9.2 analysis software as described below.
6
Thymic Epithelial Cell Analysis Protocol
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For the analysis of thymic epithelial cells (TECs), minced thymuses were digested with 1 unit/ml Liberase (Roche) in the presence of 0.01% DNase I (Roche). Single-cell suspensions were stained with antibodies specific for CD326 (EpCAM; BioLegend), CD45 (eBioscience), and CD249 (Ly51, eBioscience), and for the reactivity with UEA-1 (Vector Laboratories). For the analysis of Aire and CCL21, surface-stained cells were fixed in 4% (g/vol) paraformaldehyde, permeabilized in 0.1% saponin, and stained with anti-Aire (5H12; eBioscience) antibody or anti-CCL21 (AAM27; Bio-Rad Laboratories) antibody. For the analysis of thymocytes, splenocytes, and lymph node cells, cells were surface-stained with the indicated antibodies. For the intracellular staining of Foxp3, the surface-stained cells were fixed and permeabilized by using a Foxp3 Staining Buffer Set (eBioscience) and stained with anti-Foxp3 antibody (eBioscience). For the isolation of TECs, CD45− cells were enriched in magnetic bead conjugated anti-CD45 antibody (Miltenyi Biotec). Multicolor flow cytometry and cell sorting were performed on FACSAria II (BD).
7
Profiling Tumor Cell Signaling Pathways
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Cell lysates were prepared from HeLa, SiHa, and C33A cultures (in vitro) and from tumor cells sorted from tumors grown in mice (in vivo). Tumor single cell suspensions were obtained as described above, and CD45− cells (tumor cells) were sorted from inflammatory CD45+ cells with magnetic beads conjugated with anti-CD45 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were lysed with reagents from the Proteome Cytokine Array Kit, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). We used 100 μg of protein for each array and ECL Chemiluminescence kit (GE Healthcare, Waukesha, WI) for detection of cytokine/antibody complexes. The resulting autoradiograms were scanned for densitometry.
For Western blottings, we used 25 μg of proteins from CD45+ and CD45− cell lysates. Proteins were fractioned by SDS–Page and transferred to PVDF membranes (GE Healthcare). After blocking with 5% skim milk in 0,1% Tween 20/PBS, membranes were incubated with antibodies against anti-phosphorylated STAT3 and NFκB (p65) or anti-STAT3, NFκB and Akt (Cell Signaling Technology, Danvers, MA). Anti-tubulin was purchased from Sigma–Aldrich (St. Louis, MO). Secondary antibody was anti-rabbit from GE Healthcare, and detection was performed with ECL as described above.
For Western blottings, we used 25 μg of proteins from CD45+ and CD45− cell lysates. Proteins were fractioned by SDS–Page and transferred to PVDF membranes (GE Healthcare). After blocking with 5% skim milk in 0,1% Tween 20/PBS, membranes were incubated with antibodies against anti-phosphorylated STAT3 and NFκB (p65) or anti-STAT3, NFκB and Akt (Cell Signaling Technology, Danvers, MA). Anti-tubulin was purchased from Sigma–Aldrich (St. Louis, MO). Secondary antibody was anti-rabbit from GE Healthcare, and detection was performed with ECL as described above.
8
Single-Cell RNA Sequencing of Immune Cells
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A total of 5×106 cells were incubated with anti-CD45 antibody (Miltenyi Biotec, Inc., Cambridge, MA, USA; cat. no. 130-080-201; clone, 5B1) for 1 h at 4°C to stain white blood cells, while Hoechst 33342 (Thermo Fisher Scientific, Inc., Waltham, MA, USA; cat. no. H3570) was used to stain nuclei by adding the staining solution to the cells for 1 h at 4°C. Cells were loaded at the optimal concentration (250,000 cells/ml, as recommended by the manufacturer) into the microfluidics chip.
Single cells were isolated into individual chambers using an integrated fluidic circuit (IFC) on the Automated Microfluidic C1 system (Fluidigm Corporation, San Francisco, CA, USA). Cells positive for CD45 and Hoechst were lysed, and RNA isolation and complementary DNA (cDNA) synthesis was performed using the SMART-Seq® v4 Ultra® Low Input RNA kit for Sequencing (Clontech Laboratories, Inc., Mountainview, CA, USA; cat. no. 634888) which was preamplified using a unique SMARTer II A oligonucleotide and template switch primer (both reagents being present in the SMARTer Ultra Low RNA kit; Clontech Laboratories, Inc.; cat. no. 634833), according to the manufacturer's protocol. The cDNA was harvested manually by retrieving 3.5 µl of cDNA from the wells of the IFC for library preparation. Notably, only RNA strands with polyadenylated [poly(A)] tails were converted to cDNA and used for downstream processing.
Single cells were isolated into individual chambers using an integrated fluidic circuit (IFC) on the Automated Microfluidic C1 system (Fluidigm Corporation, San Francisco, CA, USA). Cells positive for CD45 and Hoechst were lysed, and RNA isolation and complementary DNA (cDNA) synthesis was performed using the SMART-Seq® v4 Ultra® Low Input RNA kit for Sequencing (Clontech Laboratories, Inc., Mountainview, CA, USA; cat. no. 634888) which was preamplified using a unique SMARTer II A oligonucleotide and template switch primer (both reagents being present in the SMARTer Ultra Low RNA kit; Clontech Laboratories, Inc.; cat. no. 634833), according to the manufacturer's protocol. The cDNA was harvested manually by retrieving 3.5 µl of cDNA from the wells of the IFC for library preparation. Notably, only RNA strands with polyadenylated [poly(A)] tails were converted to cDNA and used for downstream processing.
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