Wounded cell isolation was performed according to previous study [19 (link), 20 (link)]. Briefly, cells were dissociated from excisional wounds using an enzymatic digest with collagenase I, collagenase XI and hyaluronidase (Sigma Aldrich, St. Louis, MO, USA). Neutrophils, T cells, and B cells were marked by incubating cells for 15 min with fluorescein isothiocyanate-conjugated anti-Ly6G, anti-CD3, and anti-CD19. These cells were depleted from total cell population using anti-FITC magnetic beads following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes and macrophages were then positively selected using anti-CD11b magnetic beads. Cell counts were performed using hemacytometer.
Anti fitc magnetic beads
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
Anti-FITC magnetic beads are a type of lab equipment used for the isolation and separation of cells, proteins, or other biomolecules that are labeled with fluorescein isothiocyanate (FITC). These beads are coated with antibodies that specifically bind to FITC, allowing the target molecules to be captured and isolated from a complex sample.
Automatically generated - may contain errors
Lab products found in correlation
Hyaluronidase, by Merck Group (2 mentions)
Collagenase 1, by Merck Group (2 mentions)
Collagenase xi, by Merck Group (2 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Qiazol, by Qiagen (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Anti cd19 hib19, by BioLegend (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Rhil 15, by Thermo Fisher Scientific (1 mentions)
Anti cd19 6d5, by BioLegend (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Smartsec mini ev isolation system, by System Biosciences (1 mentions)
Exoel cd81a 1, by System Biosciences (1 mentions)
Sw28ti rotor, by Beckman Coulter (1 mentions)
Nucleofector kit 5, by Lonza (1 mentions)
Macs separation column, by Miltenyi Biotec (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Fitc conjugated anti mouse cd4, by BD (1 mentions)
17 protocols using anti fitc magnetic beads
1
Isolation of Wound-Infiltrating Immune Cells
2
Enrichment of Disseminated Tumor Cells
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The primary tumor and BM samples were stored in liquid nitrogen. Cryosection slides of tumors were prepared and H&E stainings were performed. Identified tumor cells‐rich regions were cut out from the respective tumor pieces and homogenized in QIAzol (Qiagen) with the gentleMACS Dissociator (Miltenyi). The dimethyl sulfoxide (DMSO) frozen MNC fraction of BM aspirates was thawed and density gradient centrifugation (LymphoprepTM, AXIS‐SHIELD PoC AS) was performed. Following the density gradient centrifugation, DTCs were labeled and enriched at 4°C as described earlier.13 In brief, the MNC fraction containing DTCs was collected after density gradient separation and washed with phosphate‐buffered saline (PBS) at 300g for 10 min at 4°C. The supernatant was discarded and the cells were resuspended in 2 ml ice‐cold magnetic‐activated cell sorting (MACS) buffer (PBS pH 7.2, 0.5% bovine serum albumin, 2 mM ethylene diamine tetraacetic acid). Thereafter, the cell suspension was incubated at 4°C for 20 min with 2.5 µl of FITC‐labeled anti‐GD2 antibodies (14.18 delta CH2 clone), followed by a 15‐min incubation with 75 µl anti‐FITC magnetic beads (Miltenyi) at 4°C. The MACS sorting was continued at 4°C and the enriched DTC fraction and the corresponding tumor cells‐depleted MNC fraction were separately collected and homogenized in QIAzol.
3
Isolation and Culture of Wound Cell Subsets
4
Isolation of Wound-Derived Cell Populations
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Cells were dissociated from human chronic wound biopsies and mouse excisional wounds using an enzymatic digest (14 (link)). Neutrophils, T cells, and B cells were marked for depletion by incubating cells for 15 min with fluorescein isothiocyanate (FITC)-conjugated anti-Ly6G (1A8), anti-CD3 (17A2), and anti-CD19 (6D5) for mouse cells and FITC-conjugated anti-CD15 (HI98), anti-CD3 (UCHT1), and anti-CD19 (HIB19) for human cells (1:10; all from Biolegend) and then depleted from the total cell population using anti-FITC magnetic beads (Miltenyi Biotec). Cells of the monocyte/Mp lineage were then isolated using CD11b magnetic beads.
5
Purification and Adoptive Transfer of Naive B Cells
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Peyer’s patch lymphocytes were isolated and stained with FITC-conjugated anti-mouse IgD (clone 11-26) (BioLegend) followed by incubation with anti-FITC magnetic beads (Miltenyi). Naive B cells were obtained by the purification of IgD-positive cells, by positive selection using MACS columns (Miltenyi). The purity was >98%. For adoptive transfer experiments, 2 × 106 of PP naive B cells were retro-orbitally transferred into Rag1−/− or JH−/− mice. Recipient mice were sacrificed 2 weeks post-transfer, and IgA production was analyzed.
6
Enrichment and Characterization of ECM1-expressing Exosomes
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High ECM1 expressing exosomes were enriched from the serum of one BCa-NTMnb subject as follows. Exosomes were incubated with a FITC labelled anti-human ECM1 antibody for 30 minutes at 4°C. The FITC-ECM1 positive exosomes were then purified using anti-FITC magnetic beads (#130-048-701, Miltenyi Biotech) followed by FACS analysis. The ECM1- exosomes from the same subject were used as a control. The HMLE cells were then co-cultured with ECM1+ and ECM1- exosome pool for 7 days [11 (link)]. The HMLE cells were cultured in media depleted of exosomes [11 (link)]. A similar experiment was performed with the exosomes isolated from one healthy and one NTMnb subject as controls.
7
Isolating Monocyte/Macrophage Lineage Cells
8
Isolation and Expansion of iNKT Cells
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Peripheral blood mononuclear cells (PBMCs) were obtained from the Immunology Core Facility at the University of Pennsylvania (Philadelphia, PA, USA) or Zenbio (Research Triangle Park, NC, USA). PBMCs were cultured in complete AIM-V media (Gibco, Waltham, MA, USA) with 10% fetal bovine serum (FBS), KRN7000 (αGC) at 500 ng/mL, and recombinant human (rh) IL-2 (Peprotech, Rocky Hill, NJ, USA) at 50 U/mL. On the fourth day, rhIL-15 (Peprotech) and rhIL-2 were added at 10 ng/mL and 10 U/mL, respectively, to the culture. After four more days, human iNKT cells were purified by staining with FITC-conjugated, anti-Vα24 antibodies, followed by anti-FITC magnetic beads, per manufacturer’s instructions (Miltenyi Biotech, Auburn, CA, USA). Post-sort FACS analysis revealed >95% PBS57–CD1d tetramer reactive cells.
9
Isolation of Wound Cell Populations
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Cells were dissociated from excisional wounds using an enzymatic digest with collagenase I, collagenase XI, and hyaluronidase [18] (link). Neutrophils, T cells, and B cells were marked for depletion by incubating cells for 15 min with fluorescein isothiocyanate (FITC)-conjugated anti-Ly6G (1A8), anti-CD3 (17A2), and anti-CD19 (6D5) (1∶10; Biolegend); these cells were depleted from the total cell population using anti-FITC magnetic beads and by following the manufacturer's instructions (Miltenyi Biotec). Cells of the monocyte/macrophage lineage were then isolated using CD11b magnetic beads. Both CD11b+ and CD11b− cell fractions were collected and then stored at −80°C for later RNA analysis. Previous studies indicated that >90% of the CD11b+ cells thus isolated were positive for monocyte/macrophage markers Ly6C and/or F4/80 by flow cytometry [18] (link). The CD11b− cell fraction likely consists mainly of fibroblasts, endothelial cells and keratinocytes.
10
Isolation and Characterization of CD47+ EVs
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EVs in mouse serum were isolated with an EV isolation kit (SmartSEC Mini EV Isolation System) and detected with an ELISA kit detecting CD81 exosome (EXOEL-CD81A-1) from System Biosciences.
L02 and primary hepatocytes (PMH) were cultured 48 hours at 37°C in serum-free DMEM. The supernatant was collected and centrifuged at 2,000 g for 10 min at 4°C to remove the cell debris [20 (link)]. Then, after filtering through 0.22 μm filters, the filtrate was ultracentrifuged at 100,000 g for 120 minutes in a Beckman SW28Ti rotor. After the resuspension by PBS, the pellets were ultracentrifuged again at 100,000 g for 120 min. The final EV pellets were dissolved in PBS for further experiments [20 (link)].
To isolate CD47+ EVs, a total of 200 μg PMH-EVs were mixed with nonblocking anti-CD47 antibody (REA170)-FITC for 30 min at 4°C. Then, incubated with anti-FITC magnetic beads (Miltenyi Biotec; 1 μL/μg EVs) overnight at 4°C. CD47− EVs and CD47+ EVs were separated by magnetic beads, and both supernatants were washed with PBS and pelleted by ultracentrifugation.
EVs were further analyzed by a Micro BCA Protein Assay Kit to determine protein concentrations (Thermo, #23235). Particle diameters and amounts were observed by the NanoSight system (NS300, Malvern, Ranch Cucamonga, CA, USA). Further details of methods were described in supplementary materials (availablehere ).
L02 and primary hepatocytes (PMH) were cultured 48 hours at 37°C in serum-free DMEM. The supernatant was collected and centrifuged at 2,000 g for 10 min at 4°C to remove the cell debris [20 (link)]. Then, after filtering through 0.22 μm filters, the filtrate was ultracentrifuged at 100,000 g for 120 minutes in a Beckman SW28Ti rotor. After the resuspension by PBS, the pellets were ultracentrifuged again at 100,000 g for 120 min. The final EV pellets were dissolved in PBS for further experiments [20 (link)].
To isolate CD47+ EVs, a total of 200 μg PMH-EVs were mixed with nonblocking anti-CD47 antibody (REA170)-FITC for 30 min at 4°C. Then, incubated with anti-FITC magnetic beads (Miltenyi Biotec; 1 μL/μg EVs) overnight at 4°C. CD47− EVs and CD47+ EVs were separated by magnetic beads, and both supernatants were washed with PBS and pelleted by ultracentrifugation.
EVs were further analyzed by a Micro BCA Protein Assay Kit to determine protein concentrations (Thermo, #23235). Particle diameters and amounts were observed by the NanoSight system (NS300, Malvern, Ranch Cucamonga, CA, USA). Further details of methods were described in supplementary materials (available
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