The expression of CD44, P63 and ABCG2 markers (as stemness markers of LESCs), was evaluated using flow cytometry. The cultured LESCs were harvested and re-suspended in PBS. Then the cells were rinsed with cold wash buffer (PBs containing 2% FBS and 0.1% sodium azide (Merck). Cells that were to be marked with surface antibodies (CD44 and ABCG2) were incubated with PE Mouse Anti-Human CD44 and APC-conjugated mouse anti-human ABCG2 (BD Pharmingen Inc) for 30 min at room temperature in dark. Cell staining with isotype-matched irrelevant antibodies (BD Pharmingen) was also performed. For intracellular staining cells were fixed with 1% cell fix for 5 minutes at 4°C and washed with 1 mL of ice-cold PBS. Then 0.2% saponin was added and incubated for 10 minutes. Then cells were centrifuged and incubated on ice with rabbit anti-human P63-α primary antibody for 1 h. Afterward, cells were washed twice with PBS and incubated with 10 μL FITC-conjugated goat anti-rabbit (Santa Cruz) secondary antibody for 30 minutes at room temperature in dark. Then cells were rinsed and analyzed with FACS CALIBUR flow cytometer (Becton Dickinson, Franklin Lakes, USA). Final data were analyzed by version 7.6 of FlowJo® software. Unstained controls were used as negative controls in the histograms to detect the percentage of positive cells.
Isotype matched irrelevant antibodies
Manufactured by BD
Isotype-matched irrelevant antibodies are a type of laboratory reagent used in immunoassays and other applications. They are antibodies that do not recognize the target antigen of interest, but share the same isotype as the primary antibody being used in the experiment. These antibodies serve as a control to help distinguish specific binding from non-specific background signals.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8, by BD (5 mentions)
Anti cd19, by BD (5 mentions)
Anti cd3, by BD (4 mentions)
Anti cd4, by BD (4 mentions)
Facsdiva software, by BD (3 mentions)
Lsrfortessa, by BD (3 mentions)
Anti cd56, by BD (3 mentions)
Anti cd45ra, by BD (2 mentions)
Anti bdca2, by Miltenyi Biotec (2 mentions)
Anti cd45ro, by BD (2 mentions)
Anti slan, by Miltenyi Biotec (2 mentions)
Ficoll hypaque, by Harvard Bioscience (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Liberase, by Roche (2 mentions)
Dnase, by Roche (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Fitc conjugated goat anti rabbit, by Santa Cruz Biotechnology (1 mentions)
Anti bdca4, by Miltenyi Biotec (1 mentions)
Anti cd38, by BD (1 mentions)
Anti hla dr, by BD (1 mentions)
11 protocols using isotype matched irrelevant antibodies
1
Evaluation of Stemness Markers in LESCs
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The expression of CD44, P63 and ABCG2 markers (as stemness markers of LESCs), was evaluated using flow cytometry. The cultured LESCs were harvested and re-suspended in PBS. Then the cells were rinsed with cold wash buffer (PBs containing 2% FBS and 0.1% sodium azide (Merck). Cells that were to be marked with surface antibodies (CD44 and ABCG2) were incubated with PE Mouse Anti-Human CD44 and APC-conjugated mouse anti-human ABCG2 (BD Pharmingen Inc) for 30 min at room temperature in dark. Cell staining with isotype-matched irrelevant antibodies (BD Pharmingen) was also performed. For intracellular staining cells were fixed with 1% cell fix for 5 minutes at 4°C and washed with 1 mL of ice-cold PBS. Then 0.2% saponin was added and incubated for 10 minutes. Then cells were centrifuged and incubated on ice with rabbit anti-human P63-α primary antibody for 1 h. Afterward, cells were washed twice with PBS and incubated with 10 μL FITC-conjugated goat anti-rabbit (Santa Cruz) secondary antibody for 30 minutes at room temperature in dark. Then cells were rinsed and analyzed with FACS CALIBUR flow cytometer (Becton Dickinson, Franklin Lakes, USA). Final data were analyzed by version 7.6 of FlowJo® software. Unstained controls were used as negative controls in the histograms to detect the percentage of positive cells.
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The expression of CD44, P63 and ABCG2 markers (as stemness markers of LESCs), was evaluated using flow cytometry. The cultured LESCs were harvested and re-suspended in PBS. Then the cells were rinsed with cold wash buffer (PBs containing 2% FBS and 0.1% sodium azide (Merck). Cells that were to be marked with surface antibodies (CD44 and ABCG2) were incubated with PE Mouse Anti-Human CD44 and APC-conjugated mouse anti-human ABCG2 (BD Pharmingen Inc) for 30 min at room temperature in dark. Cell staining with isotype-matched irrelevant antibodies (BD Pharmingen) was also performed. For intracellular staining cells were fixed with 1% cell fix for 5 minutes at 4°C and washed with 1 mL of ice-cold PBS. Then 0.2% saponin was added and incubated for 10 minutes. Then cells were centrifuged and incubated on ice with rabbit anti-human P63-α primary antibody for 1 h. Afterward, cells were washed twice with PBS and incubated with 10 μL FITC-conjugated goat anti-rabbit (Santa Cruz) secondary antibody for 30 minutes at room temperature in dark. Then cells were rinsed and analyzed with FACS CALIBUR flow cytometer (Becton Dickinson, Franklin Lakes, USA). Final data were analyzed by version 7.6 of FlowJo® software. Unstained controls were used as negative controls in the histograms to detect the percentage of positive cells.
2
Comprehensive Immune Cell Profiling
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Freshly prepared PBMCs were used. Subpopulations of T cells, B cells, natural killer (NK) cells and antigen-presenting cells (APC) were characterized by surface staining with fluorescence labelled anti-CD3, anti-CD4, anti-CD5, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD25, anti-CD27, anti-CD38, anti-CD45RA, anti-CD45RO, anti-CD56 and anti-HLA-DR (BD Bioscience); anti-BDCA1, anti-BDCA2, anti-BDCA3, anti-BDCA4 and anti-slan (Miltenyi Biotec). Negative controls included directly labeled or unlabeled isotype-matched irrelevant antibodies (BD Biosciences). Freshly prepared CSF cells were used directly for FACS analysis. Fluorescence-labeled antibodies for surface staining were used as follows: anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD25, anti-CD27, anti-CD45RA, anti-CD45RO, anti-CD56 and anti-HLA-DR (BD Bioscience); anti-BDCA1 and anti-slan (Miltenyi Biotec). All cells were measured on a LSR-Fortessa (BD Biosciences) and evaluated by FACS-Diva Software (BD Bioscience).
3
Standardized Blood Cell Phenotyping
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Standardized blood testing was performed for complete blood cell counts and peripheral cell subsets at the Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Dresden, Germany. Immune cell phenotyping was done by flow cytometry using fluorescence-labeled anti-CD3, anti-CD4, anti-CD8, and anti-CD19 (BD Biosciences, Heidelberg, Germany) according to the manufacturer’s instructions and evaluated on FACSCanto II. Negative controls included directly labeled or unlabeled isotype-matched irrelevant antibodies (BD Biosciences).
4
TLR2 and TLR4 Expression Analysis
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For TLR2 staining, 5×105 living cells were incubated with 5 μg/ml of Alexa fluor 488-conjugated anti-TLR2 antibody (BD) diluted in PBS-BSA for 30 min. All incubations were performed on ice. For TLR4 staining, cells were first incubated with biotin-conjugated anti-TLR4 (BD) for 60 min. After three washes, cells were stained with PE-Cy5.5-conjugated streptavidin (Invitrogen, USA) for 30 min. In negative reagent control tubes, isotype matched irrelevant antibodies (BD) were used instead of primary antibodies. Monocyte gate of PBMC served as the positive control. After three washing steps with PBS-BSA, cells were analyzed by Partec flowcytometer (Partec, Germany).
5
Characterizing Immune Cell Subsets
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After blood collection, subpopulations of T-cells, B-cells, and natural killer (NK) cells were characterized by surface staining with fluorescence labeled anti-CD3, anti-CD4, anti-CD8, anti-CD16, anti-CD14, anti-CD19, anti-CD56 (BD Biosciences, Heidelberg, Germany) according to the manufacturer's instructions. Negative controls included directly labeled or unlabeled isotype-matched irrelevant antibodies (BD Biosciences). Cells were evaluated on FACSCanto II flow cytometer.
6
Flow Cytometric Gating of Nucleated Cells
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Staining of nucleated cells was determined as before (9 (link), 19 (link)) by gating on forward- and side-scatter properties with isotype-matched irrelevant antibodies (PharMingen) as negative controls. Ten thousand viable counts were analyzed with a FACScan flow cytometer that was standardized with SpheroParticles (Spherotech Inc., Chicago, IL) and CELLQUEST software (Becton Dickinson, San Jose, CA).
7
Analyzing Immune Cell Populations in CSF
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Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll‐Hypaque (Biochrom, Berlin, Germany) density centrifugation. CSF samples were centrifuged at 250 g for 10 min at 4°C within 20 min after collection, and the cell pellet was immediately processed by flow cytometry. Cell surface staining was performed using fluorescence labeled anti‐CD3, anti‐CD4, anti‐CD8, anti‐CD19, anti‐CD45RA, anti‐CD45RO and anti‐CD138 antibodies (BD Biosciences, Heidelberg, Germany) according to the manufacturer´s instructions. Negative controls included directly labeled isotype‐matched irrelevant antibodies (BD Biosciences). After the staining procedure, cells were evaluated by flow cytometry. Cells were measured on a LSR‐Fortessa (BD Biosciences) and evaluated by FACS‐Diva Software (BD Bioscience).
8
Immunophenotyping of PBMC Subsets
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After blood collection, peripheral blood mononuclear cells (PBMC) were prepared by Ficoll-Hypaque (Biochrom, Berlin, Germany) density centrifugation. Subpopulations of T cells, B cells, natural killer (NK) cells, and antigen-presenting cells (APC) were characterized by surface staining with fluorescence-labeled anti-CD3, anti-CD4, anti-CD8, anti-CD16, anti-CD14, anti-CD19, anti-CD56 (BD Biosciences, Heidelberg, Germany) or anti-BDCA2, and anti-slan (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer's instructions. Negative controls included directly labeled or unlabeled isotype-matched irrelevant antibodies (BD Biosciences). Cells were evaluated on LSR-Fortessa (BD Biosciences).
9
Renal cell isolation and flow cytometry
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Renal cell isolation and flow cytometry were performed as described11 (link). Briefly, kidneys were minced and incubated at 37°C for 30 minutes in PBS containing 0.25 mg/ml Liberase TM (Roche) and 10 U/ml Dnase (Roche). Cells were filtered through a 40μm strainer, washed, centrifuged and resuspended in FACS buffer. Cells (5 × 105) were first incubated with FITC-anti-CD45 (BD Biosciences, San Jose, CA), and goat-anti-DDR2 (Santa Cruz Biotech, Santa Cruz, CA) followed by followed by anti-goat-APC (BD Biosciences, San Jose, CA). Cells incubated with irrelevant isotype-matched antibodies (BD Biosciences, San Jose, CA) and unstained cells were used as controls. The cutoffs were set according to results of controls. The fluorescence intensities were measured using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using BD FACSDiva software.
10
Multilineage Characterization of hMSCs
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ISL1-hMSCs or Ctrl-hMSCs were analyzed by flow cytometry with primary fluorescence using fluorescein isothiocyanate (FITC)-conjugated (anti-CD29, anti-CD34, and anti-CD45) or phycoerythrin (PE)-conjugated (anti-CD14, anti-CD31, anti-CD73, and anti-CD90) antibodies (BD Biosciences, San Diego, CA). Irrelevant isotype-matched antibodies (BD Biosciences, San Diego, CA) were used as negative controls. Flow cytometry analyses were performed with an Influ cell sorter (BD Biosciences, San Diego, CA). The data were analyzed using FlowJo7.5 software (Treestar, Ashland, OR, USA).
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