NHDFs were isolated from cocultures by using a magnetic activated cell separation (MACS) system with anti-Fibroblast MicroBeads (130-050-601, Miltenyi Biotec, Auburn, CA, USA) and an MS column (130-042-201, Miltenyi Biotec) according to the manufacturer’s protocol.
Anti fibroblast microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
Anti-fibroblast microbeads are a laboratory product designed for the separation and isolation of fibroblasts from cell samples. The core function of these microbeads is to facilitate the efficient and targeted removal of fibroblasts from heterogeneous cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 t cell isolation kit, by Miltenyi Biotec (3 mentions)
Trypsin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (3 mentions)
Collagenase, by Merck Group (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
Interleukin 2 (il 2), by Roche (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Collagenase 1, by Worthington (2 mentions)
Quadromacs separator, by Miltenyi Biotec (2 mentions)
Collagenase p, by Roche (2 mentions)
Facscan, by BD (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Anti cd45 microbeads, by Miltenyi Biotec (1 mentions)
Macs separator, by Miltenyi Biotec (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
35 protocols using anti fibroblast microbeads
1
Isolation of Fibroblasts from Co-cultures
2
Isolation and Characterization of CD8+ T Cells
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Following removal of dead cells, CD8+ T cells were isolated using negative magnetic bead selection with the CD8+ T cell isolation kit (Miltenyi Biotec) following instructions with minor modifications. This negative selection protocol delivers untouched CD3+CD8+ T cells. Additionally, anti-fibroblast microbeads (Miltenyi Biotec) were added in combination with the microbeads supplied with the kit to ensure depletion of stromal fibroblasts present in the mixed cell suspension as described before for CD4 selection (3 (link)). After two rounds of negative selection, purity of the CD8+ T cell population was higher than 90%, with ~2% contamination with non-immune cells, 2% CD3- cells and 1–2% contamination with CD4+ T cells (Supplementary Figure 1A ). Following isolation, CD8+ T cells were used in cytotoxicity assays without in vitro stimulation, or stimulated for degranulation and proliferation assays.
3
Isolation and Purification of Vascular Endothelial Cells
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The isolated cells were sorted by the Miltenyi Biotech magnetic sorting system as shown in Figure 1 . The first step was to add Anti-CD45 MicroBeads and Anti-Fibroblast MicroBeads (the latter are conjugated to monoclonal mouse anti-human fibroblast antibodies and mainly target CD44) (Miltenyi Biotech) to the cells and to incubate the mixture at room temperature for 10 min. Anti-CD31-PE antibodies (Miltenyi Biotech) were then added, followed by incubation at 4°C in the dark. Subsequently, the cell suspension was subjected to magnetic sorting using LS Columns (Miltenyi Biotech) that had been washed three times with PEB. The cells that passed through the column were collected and centrifuged (300 g, 10 min, 4°C), after which the supernatant was removed. Anti-PE-MicroBeads (Miltenyi Biotech) and PEB were then added to the cells and mixed well. The resulting cell suspensions were loaded onto thrice-washed LS Columns and the cells that passed through were discarded. After removing the column from the magnet, the cells were washed out into a tube and then, to increase the purity of the VECs, passed through another LS column. Thereafter, the resulting suspension was immediately placed in RNAlater (Thermo Fisher Scientific). Some of the suspension was analyzed by flow cytometry using FACScan (BD Biosciences, Franklin Lakes, NJ, United States) to determine the purity of the VECs.
4
Isolation and Purification of hCPCs
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To isolate hCPCs, myocardial specimens were dissected, minced, and enzymatically disaggregated as previously described (Nurzynska et al., 2013 (link)). Briefly, samples were digested with 0.25% trypsin and 0.1% collagenase (both from Sigma-Aldrich) and the obtained cell population was depleted of fibroblasts by incubation with anti-fibroblast MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), to magnetically label fibroblasts, and then loading cells onto a MACS column (Miltenyi Biotec) placed in the magnetic field of a MACS separator (Miltenyi Biotec) to retain labeled fibroblasts within the column and allow unlabeled cells to run through. Hence, hCPCs were purified from the negative cell fraction by positive selection with anti-human-CD117 MicroBeads. The so obtained hCPCs were then collected and used for repopulating d-HuSk and d-HuM to evaluate d-HuSk cytocompatibility and ability to support hCPC engraftment and differentiation potential.
5
Isolation of Synovial Leukocytes from RA Patients
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Synovial biopsies from RA patients undergoing surgery were obtained and leucocyte isolation was performed as follows. Tissue was cut into pieces and incubated with an enzyme solution (collagenase, hyaluronidase, DNAse in RPMI) for 90 min and 37° under constant rotation. Single cell suspension was obtained using gauze and smooth mechanical disruption of digested tissue. Subsequently cells were sorted for non-fibroblasts using anti-fibroblast microbeads from Miltenyi. Non-fibroblast were used for FACS analysis and CD45 staining was used additionally to other antibodies in order to discriminate non-leucocytes.
6
Isolation and Culture of Tumor Cells
7
Isolation and Activation of CD4+ T Cells
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Following removal of dead cells, CD4+ T cells were isolated by negative magnetic bead selection with the CD4+ T cell isolation kit (Miltenyi Biotec) following instructions with minor modifications, as previously described35 (link). Anti-fibroblast microbeads (Miltenyi Biotec) were added in combination with the microbeads supplied with the kit to ensure depletion of stromal fibroblasts present in the mixed cell suspension. After two rounds of negative selection, purity of the CD4+ T cell population was higher than 90%. Isolated CD4+ T cells were activated for 24 hr with PHA and IL-2 as described for blood CD4+ T cells. Activated CD4+ T cells were plated at a density of 1 × 105 cells per well in round-bottom ultra-low attachment 96-well culture plates (Corning, Corning, NY) in 0.2 ml of Immune Cell Media.
8
CD4+ T Cell Isolation and Activation
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Following removal of dead cells, CD4+ T cells were isolated using negative magnetic bead selection with the CD4+ T cell isolation kit (Miltenyi Biotec) following instructions with minor modifications. This negative selection process depletes CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ/δ and CD235a and delivers untouched CD3+CD4+ T cells. Additionally, anti-fibroblast microbeads (Miltenyi Biotec) were added in combination with the microbeads supplied with the kit to ensure depletion of stromal fibroblasts present in the mixed cell suspension. After two rounds of negative selection, purity of the CD4+ T cell population was higher than 90% (see Figure 2A ). Yield recovery of CD4+ T cells is shown in Supplementary Figure 1b . Following isolation, cells were analyzed by RT-PCR as described below, without in vitro stimulation.
CD4+ T cells from blood were isolated with the CD4+ T cell isolation kit (Miltenyi Biotec) following standard ficoll centrifugation, and activated in vitro with Phytohemagglutinin (PHA) (2.5 µg/ml; Sigma, St Louis, MO) and IL-2 (50 U/ml) (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann- La Roche Inc)38 (link) for 24h as described before8 (link).
CD4+ T cells from blood were isolated with the CD4+ T cell isolation kit (Miltenyi Biotec) following standard ficoll centrifugation, and activated in vitro with Phytohemagglutinin (PHA) (2.5 µg/ml; Sigma, St Louis, MO) and IL-2 (50 U/ml) (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann- La Roche Inc)38 (link) for 24h as described before8 (link).
9
Isolation of Cytotrophoblast Cells from First Trimester Placenta
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We have successfully isolated cytotrophoblast cells from human term placentas [31 (link)]. Here, we isolated cytotrophoblast cells from
human first trimester placentas using similar methods with minor modifications. Briefly,
villous tissues were dissected free of membranes, rinsed, and minced in phosphate-buffered
saline (PBS) (Life Technologies). The villous samples were digested three times in a
digestion enzyme medium containing 1 mg/ml Dispase II (Life Technologies) and 0.1 mg/ml
DNase I (Roche) at 37°C for 15 min each cycle. Released cells were then purified on a
discontinuous Percoll gradient (GE Healthcare) and centrifuged at 730 × gfor 20 min at 4°C. The layer between the 45% and 35% Percoll aliquots containing
cytotrophoblast cells (density: 1.050–1.060 g/ml) were collected. Collected cells were
further immunopurified by eliminating CD45RB-positive cells of myeloid origin using a
phycoerythrin (PE)-conjugated anti-CD45RB antibody (Ab) (1:10; Miltenyi Biotec) and
anti-PE-microbeads (1:5; Miltenyi Biotec), and depleting fibroblasts using anti-fibroblast
microbeads (1:5; Miltenyi Biotec) according to the manufacturer's instructions.
human first trimester placentas using similar methods with minor modifications. Briefly,
villous tissues were dissected free of membranes, rinsed, and minced in phosphate-buffered
saline (PBS) (Life Technologies). The villous samples were digested three times in a
digestion enzyme medium containing 1 mg/ml Dispase II (Life Technologies) and 0.1 mg/ml
DNase I (Roche) at 37°C for 15 min each cycle. Released cells were then purified on a
discontinuous Percoll gradient (GE Healthcare) and centrifuged at 730 × gfor 20 min at 4°C. The layer between the 45% and 35% Percoll aliquots containing
cytotrophoblast cells (density: 1.050–1.060 g/ml) were collected. Collected cells were
further immunopurified by eliminating CD45RB-positive cells of myeloid origin using a
phycoerythrin (PE)-conjugated anti-CD45RB antibody (Ab) (1:10; Miltenyi Biotec) and
anti-PE-microbeads (1:5; Miltenyi Biotec), and depleting fibroblasts using anti-fibroblast
microbeads (1:5; Miltenyi Biotec) according to the manufacturer's instructions.
10
Isolation and Culture of FMT Organoids
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Three FMT lesions, collected immediately after surgical removal, were obtained from the Nyan cat clinic, An-Sing animal hospital and the veterinary medical teaching hospital of National Chung-Hsing University. Solid FMT tissue was cut into small pieces using a scalpel or scissors. The tissue was digested into organoids with digestion buffer (2 mg/ml Type 3 collagenase (Worthington) and 100 U/ml hyaluronidase (Sigma) in DMEM medium) in a 200 rpm shaker at 37 °C overnight. The organoids were digested by TrypLE (Gibco) for 10 min at 37 °C. The isolated cells were cultured in DMEM medium containing 10% fetal bovine serum. FMT cells were immunomagnetically deprived with anti-fibroblast microbeads (Miltenyi Biotec) to avoid fibroblast contamination.
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