The antigenic phenotype of UCB cells was assessed before and during ex vivo expansion. Approximately 1 × 105 cells per test were incubated with the required antibody panel as per manufacturer’s instructions for 30 min at room temperature. All antibodies were preconjugated and monoclonal. Progenitor panel: CD34-PerCP-Vio700, CD38-FITC, CD45RA-VioGreen, CD90-APC-Vio770, CD133-APC, CD135-PE-Vio770 (Miltenyi Biotech) and CD33-PE (BD Biosciences). Mature stage myeloid panel: CD13-APC, CD14-PE, CD15-VioGreen, CD34-PerCP-Vio700, CD33-APC-Vio770, CD38-FITC, CD123-VioBlue (Miltenyi Biotech). Analysis was conducted with CITRUS (cluster identification, characterisation and regression) software [31 (link)]. The software uses a regularized supervised learning algorithm to determine the populations and features of the samples being analysed in a correlative manner. The results of a CITRUS run are clusters (populations) that differentiate the observed endpoint of the samples, and the features (relative population abundance or median expression of a functional marker) of the clusters that are responsible. All CITRUS analyses were conducted using a Significance Analysis of Microarrays (SAM) association model and a minimum cluster size of 4%, at least 5000 events from each sample were included for the analysis.
Cd38 fitc
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
CD38-FITC is a fluorescent-labeled antibody that binds to the CD38 antigen, a transmembrane glycoprotein expressed on various cell types. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 pe, by Miltenyi Biotec (2 mentions)
Facslyric flow cytometer, by BD (2 mentions)
Facssuite, by BD (2 mentions)
Anti igd vioblue, by Miltenyi Biotec (2 mentions)
Cd21 apc vio770, by Miltenyi Biotec (2 mentions)
Anti igm pe, by Miltenyi Biotec (2 mentions)
Cd27 apc, by Miltenyi Biotec (2 mentions)
Cd86 pe vio770, by Miltenyi Biotec (2 mentions)
Cd24 percp vio700, by Miltenyi Biotec (2 mentions)
Cd19 viogreen, by Miltenyi Biotec (2 mentions)
Cd33 pe, by BD (1 mentions)
Cd14 pe, by Miltenyi Biotec (1 mentions)
Cd133 apc, by Miltenyi Biotec (1 mentions)
Cd90 apc vio770, by Miltenyi Biotec (1 mentions)
Cd45ra viogreen, by Miltenyi Biotec (1 mentions)
Cd13 apc, by Miltenyi Biotec (1 mentions)
Cd34 percp vio700, by Miltenyi Biotec (1 mentions)
Cd33 apc vio770, by Miltenyi Biotec (1 mentions)
Cd44 fitc, by Bio-Rad (1 mentions)
Igg1 fitc, by Miltenyi Biotec (1 mentions)
6 protocols using cd38 fitc
1
Immunophenotyping of Expanded UCB Cells
2
Immunophenotyping of Mesenchymal Stem Cells
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Immunophenotyping of MSC by flow cytometry was reported elsewhere[14 (link)]. Unless stated otherwise, fluorescence-conjugated monoclonal antibodies from Beckman Coulter were used. They were IgG1-FITC, IgG1-PE, HLA-DR-FITC, CD45-FITC, CD3-FITC, CD19-PE, CD16-FITC, CD33-FITC, CD38-FITC, CD34-PE, CD133-PE (Miltenyi Biotec GmbH, Germany), CD29-PE, CD44-FITC, CD73-FITC, CD90-PE, CD105-PE (Serotec, United Kingdom) and CD166-PE were used. At least 10000 events were acquired and signals were analysed by using the Coulter Epic XL MCL flow cytometer (Coulter, Miami, FL, United States).
Procedural details of immunofluorescence staining were described previously[15 (link)]. IgM anti-stage-specific embryonic antigen-4 (SSEA-4, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, United States), IgG2b anti-octamer-binding transcription factor-4 (Oct-4; 1:100, Santa Cruz Biotechnology), IgG1 anti-Nestin (1:400; BD Biosciences, San Francisco, CA, United States) were employed.
Cell viability was evaluated by using trypan blue dye exclusion test. Sterility check against microbial contamination was conducted at each MSC passage.
Procedural details of immunofluorescence staining were described previously[15 (link)]. IgM anti-stage-specific embryonic antigen-4 (SSEA-4, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, United States), IgG2b anti-octamer-binding transcription factor-4 (Oct-4; 1:100, Santa Cruz Biotechnology), IgG1 anti-Nestin (1:400; BD Biosciences, San Francisco, CA, United States) were employed.
Cell viability was evaluated by using trypan blue dye exclusion test. Sterility check against microbial contamination was conducted at each MSC passage.
3
Flow Cytometric Analysis of Hematopoietic Cells
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PBMCs, PB-CD34+ cells, and BM-CD34+ cells were tested for cell surface antigen expression by immunofluorescence and flow cytometric analysis. Cells were harvested and rinsed with PBS, following which CD34-PE (Miltenyi Biotec, #130-113-179, 1:50), CD38-FITC (Miltenyi Biotec, #130-113-426, 1:50), CD45RA-APC (Miltenyi Biotec, #130-117-742, 1:50), and 7-AAD (eBioscience, Waltham, MA, USA, 3 μL/105 cells) were added. Thereafter, cells were incubated at 4 °C for 10 min in the dark. Finally, the stained cells were analyzed using a BD AccuriTM C6 (BD Biosciences).
4
Immunophenotyping of Lymphocytes and B-cells
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Blood samples from the baseline visit were processed within 4 hours for analysis by flow cytometry. For lymphocyte phenotyping, ethylenediaminetetraacetic acid whole blood was stained for CD3, CD4, CD8, CD45, CD16, CD56, and CD19, as previously described (30 (link)). For B-cell phenotyping, PBMCs were isolated from lithium heparin whole blood by Ficoll gradient density centrifugation. One million PBMCs were incubated with the following antibodies: CD19-VioGreen, anti-IgD-VioBlue, CD24-PerCP-Vio700, CD38-FITC, CD27-APC, CD86-PE-Vio770, CD21-APC-Vio770, and anti-IgM-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were measured using a FACSLyric flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using the FACSSuite (BD Biosciences). The gating strategy is shown in Supplemental Figure 1
5
Comprehensive Immune Cell Profiling
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Blood samples from the baseline visit were processed within 4 h for analysis by flow cytometry. For lymphocyte phenotyping, ethylenediaminetetraacetic acid whole blood was stained for CD3, CD4, CD8, CD45, CD16, CD56, and CD19. For immune cell phenotyping, PBMCs were isolated from lithium heparin whole blood by Ficoll gradient density centrifugation. One million PBMCs were incubated with the following antibodies: CD19-VioGreen, anti-IgD-VioBlue, CD24-PerCP-Vio700, CD38-FITC, CD27-APC, CD86-PE-Vio770, CD21-APC-Vio770, and anti-IgM-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were measured using a FACSLyric flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using FACSSuite (BD Biosciences).
6
Evaluating Immune Activation Markers
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Immune activation was evaluated based on the percentage of CD4+ and CD8+ lymphocytes expressing CD38 and major histocompatibility complex class II (HLA-DR) molecules [31 (link)]. The different CD4+-cell subsets were identified using cell-surface markers: CXCR3+CCR6−CD4+ (“Th1”), CXCR3−CCR6−CD4+ (“Th2”), CXCR3−CCR6+CD4+ (“Th17”) [32 (link)], and CD25highCD4+ (“Treg”) [33 (link)].
Blood samples were processed for cell staining within 6 h after collection. Whole blood samples (50 µL) were stained for 15 min at 4 °C with a combination of four monoclonal antibodies: anti-CD4 PerCP and CD8 PE (BD Biosciences, San Jose, CA, USA), CD38 FITC (Miltenyi Biotec, Auburn, AL, USA), and HLA-DR PE-Cy7 (Biolegend, San Diego, CA, USA). Alternatively, samples were stained with a combination of four anti-CD4 monoclonal antibodies: anti-CD4 PerCP and CD25 PE (BD Biosciences), and CXCR3 FITC and CCR6 PE-Cy7 (Biolegend). After red blood cells were lysed with a lysing buffer (BD Biosciences), the remaining cells were washed once with 1 mL of phosphate buffered saline (PBS), kept at 4 °C in PBS with 1% paraformaldehyde and 0.5% bovine serum albumin, and analyzed within 48 h after staining at Kanazawa University with a JSAN flow cytometer (Bay Bioscience, Kobe, Japan). The data were analyzed with Flowjo V.7.5.5 (FLOWJO, OR, USA).
Blood samples were processed for cell staining within 6 h after collection. Whole blood samples (50 µL) were stained for 15 min at 4 °C with a combination of four monoclonal antibodies: anti-CD4 PerCP and CD8 PE (BD Biosciences, San Jose, CA, USA), CD38 FITC (Miltenyi Biotec, Auburn, AL, USA), and HLA-DR PE-Cy7 (Biolegend, San Diego, CA, USA). Alternatively, samples were stained with a combination of four anti-CD4 monoclonal antibodies: anti-CD4 PerCP and CD25 PE (BD Biosciences), and CXCR3 FITC and CCR6 PE-Cy7 (Biolegend). After red blood cells were lysed with a lysing buffer (BD Biosciences), the remaining cells were washed once with 1 mL of phosphate buffered saline (PBS), kept at 4 °C in PBS with 1% paraformaldehyde and 0.5% bovine serum albumin, and analyzed within 48 h after staining at Kanazawa University with a JSAN flow cytometer (Bay Bioscience, Kobe, Japan). The data were analyzed with Flowjo V.7.5.5 (FLOWJO, OR, USA).
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