Single dissociated Ishikawa and Hec-1a cells were suspended in buffer composed of 5% (v/v) bovine serum albumin (BSA), 2mM ethylenediaminetetracetic acid (EDTA) and PBS and incubated with either CD133-APC or CD133-VioBright FITC antibodies (both monoclonal mouse, Miltenyi Biotec, dilution 1:500) and FcR blocking reagent (Miltenyi Biotec, dilution 1:5) or isotype control antibody (mouse IgG1, Miltenyi Biotec, dilution 1:11) for 10 min in the dark at 2–8 °C. Following incubation, cells were washed and resuspended in the buffer for flow cytometry.
Mouse igg1
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
Mouse IgG1 is an immunoglobulin G1 (IgG1) isotype antibody derived from mouse. It is a common laboratory reagent used in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (2 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Human progranulin duoset elisa, by R&D Systems (1 mentions)
96 well plate, by Corning (1 mentions)
Moflow flow cytometer, by Beckman Coulter (1 mentions)
Mouse igg1, by Thermo Fisher Scientific (1 mentions)
Rat igg2a, by Miltenyi Biotec (1 mentions)
Influx flow cytometer, by BD (1 mentions)
Sytox blue, by Thermo Fisher Scientific (1 mentions)
Easysep pe selection kit, by STEMCELL (1 mentions)
Facsaria 3, by BD (1 mentions)
Mouse igg2a, by BD (1 mentions)
8 protocols using mouse igg1
1
Identification of CD133+ cells
2
Evaluating Progranulin Levels in Glioblastoma Cells
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U251 human glioblastoma cells (JCRB) were seeded at a density of 1 × 104 cells/well in a 96-well plate (Corning) in 100 μL of growth media MEM with Glutamax (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin combination (FUJIFILM Wako Pure Chemical). The cells were incubated at 37°C and 5% CO2 for 24 h and treated with the various concentrations of mAbs or PBS and incubated at 37°C and 5% CO2 for 72 h. The isotype control antibody used was mouse IgG1 (Miltenyi Biotec). Cell supernatant was collected, and ELISA was performed using Human Progranulin DuoSet ELISA (R&D Systems) as per the manufacturer’s instructions. OD450 was measured with the ARVO plate reader. Progranulin concentration was normalized against PBS-treated cells to identify relative changes in the progranulin levels.
3
Isolation and Characterization of Stromal Cells
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Isolated stromal cells (up to 1 x 107 cells/100 μl) were incubated with antibody combinations of allophycocyanin (APC)-conjugated CD49f (1:10, clone GoH3, rat IgG2a; Miltenyi Biotec) and Alexa Fluor 488-conjugated CD45 (1: 20; mouse IgG1; Life Technologies) or phycoerythrin (PE)-conjugated CD271 (1:10, mouse IgG1; Miltenyi Biotec) and APC-conjugated CD49f in 2% fetal bovine serum/PBS (FBS/PBS) for 30 min on ice in the dark. Cells were then washed and resuspended in 1 μM Sytox Blue to distinguish live and dead cells (Life Technologies) and fluorescence activated cell sorting (FACS) was undertaken on a MoFlow flow cytometer (Beckman Coulter) or an Influx flow cytometer (Becton Dickinson Biosciences).
4
Isolation and Transplantation of CD133+ Liver Progenitor Cells
5
Quantifying HER Protein Expression
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Levels of cell surface expression of HER proteins were evaluated at the University of Michigan Cancer Center core facility on a Fortesse Cell analyzer (Becton Dickinson Biosciences, San Jose, CA) using FAC Diva 8.0.1 software (Becton Dickinson Biosciences, San Jose, CA). The following antibodies were used: Phycoerythrin or fluorescein isothiocyanate labeled rat monoclonal antibody to human EGFR (ICR 10, Abcam, Eugene, OR); allophycocyanin (APC) labeled anti-HER2 (Becton Dickinson Biosciences, San Jose, CA); APC labeled anti-human HER3 (R&D Systems, Minneapolis, MN); APC labeled anti-human HER4 (R&D Systems, Minneapolis, MN), and isotype controls mouse IgG2A (R&D Systems, Minneapolis, MN) and mouse IgG1 (Miltenyi Biotech, San Diego, CA). Cells were incubated with antibodies for 30 minutes-1 hour in the dark at room temperature on a rotator. After incubation, the cells were washed with phosphate buffered saline containing 1% fetal bovine serum (1% FBS/PBS), centrifuged, and resuspended in 1% FBS/PBS. The samples were kept on ice in the dark prior to analysis on the flow cytometer.
6
CD133 Expression in HPAF-II Cells
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The expression levels of CD133 in the HPAF-II cell line were assessed by flow cytometry with an anti-CD133/2-phycoerythrin (PE) monoclonal antibody (MAb) [#130-080-901; mouse immunoglobulin (IgG)1; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany]. A mouse IgG1 MAb served as a control (#130-092-212; Miltenyi Biotec GmbH).
To perform flow cytometry analysis, cells were trypsinized when 80% of confluence was reached. For each analysis, 5×105 cells were used. Cells were incubated with a mouse IgG1 MAb solution (1:80) for 10 min at 4°C, and next resuspended in an anti-CD133/2-PE antibody solution (1:10) for 10 min at 4°C in the dark. Upon incubation, the cells were washed with 0.1% PBS two times, and resuspended in 500 µl magnetic-activated cell sorting (MACS) buffer [phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA)], prior to be analyzed in a FACSCalibur™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). A cell suspension that was only incubated with mouse IgG1 MAb was used as a control. Analysis of the results was performed using FlowJo version 7.2.5 software (FlowJo, LLC, Ashland, OR, USA).
To perform flow cytometry analysis, cells were trypsinized when 80% of confluence was reached. For each analysis, 5×105 cells were used. Cells were incubated with a mouse IgG1 MAb solution (1:80) for 10 min at 4°C, and next resuspended in an anti-CD133/2-PE antibody solution (1:10) for 10 min at 4°C in the dark. Upon incubation, the cells were washed with 0.1% PBS two times, and resuspended in 500 µl magnetic-activated cell sorting (MACS) buffer [phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA)], prior to be analyzed in a FACSCalibur™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). A cell suspension that was only incubated with mouse IgG1 MAb was used as a control. Analysis of the results was performed using FlowJo version 7.2.5 software (FlowJo, LLC, Ashland, OR, USA).
7
Quantifying Surface Proteins in CAFs
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Surface expression of TGFβRII and PDGFRβ on control and irradiated CAFs (IR‐CAFs) was examined by flow cytometry on BD FACSAria III instrument, and by the FlowJo software, Ver.7.2.4 (Tree Star‐USA). In short, CAF/IR‐CAF cells (2.5 × 105 cells/condition) were immuno‐labeled (30 min at 4°C) with several fluorescent antibodies specific for each distinct surface marker (Table 4 ). Isotype controls included REA control (#130‐104‐612; Miltenyi Biotec), mouse IgG1 (#130‐098‐845; Miltenyi Biotec), and mouse IgG2a (#555574; BD Biosciences‐USA).
8
CD133 Expression in Renal Cancer Cells
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786-O and ACHN adherent cells and tumorpheres were treated with selective concentrations of CSE for 5 days respectively, and cells were collected and washed by ice-cold PBS twice. Afterwards, 1 × 106 cells were exposed to 1 μL APC-conjugated human monoclonal CD133/1(AC133) (Miltenyi Biotech, Teterow, Germany) antibody or isotype control antibody (Mouse IgG1) (Miltenyi Biotech) at 4 °C in the darkness for 10 min subsequently re-suspended in 400 μl PBS and detected by flow cytometry analysis.
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