Following crypt isolation, the crypt suspensions were pelleted (100 g, 5 min, 4°C) and supernatant was discarded. Crypts were resuspended in TrypLE (GIBCO, no phenol red, 12604039) and dissociated into individual cells by warming crypts in water bath at 32°C for 60 s. Dissociated single cells were treated with the following antibody cocktail for flow cytometry analysis: CD45-PE (eBioscience, 12-0451-83), CD31-PE (Biolegend, 102514), Ter-119PE (Biolegend, 116208), CD24-Pacific Blue (Biolegend, 101820), CD117-APC/Cy7 (Biolegend, 105826), and EpCAM-APC (eBioscience, 17-5791-82). 7AAD (Invitrogen, A1310) was used a viability dye to exclude dead cells from the analysis. ISCs were isolated as Lrg5-eGFPhiEpCAM+CD24low/−CD31−Ter119−CD45−7AAD−. eGFPlow progenitors were isolated as Lrg5-eGFPlowEpCAM+CD24low/−CD31−Ter119−CD45−7AAD−. Cells were sorted using a BD FACS II SORP cell sorter.
Cd117 apc cy7
Manufactured by BioLegend
Sourced in United States
The CD117-APC/Cy7 is a fluorochrome-conjugated antibody that binds to the CD117 antigen, also known as c-Kit. CD117 is a tyrosine kinase receptor that plays a crucial role in the development and proliferation of various cell types, including hematopoietic stem cells, mast cells, and germ cells. This antibody can be used for the identification and analysis of CD117-expressing cells in flow cytometry applications.
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Lab products found in correlation
Sca 1 pe cy7, by BioLegend (3 mentions)
7 aad, by Thermo Fisher Scientific (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Epcam apc, by Thermo Fisher Scientific (2 mentions)
Cd45 pe, by Thermo Fisher Scientific (2 mentions)
Cd31 pe, by BioLegend (2 mentions)
Ter119 pe, by BioLegend (2 mentions)
Cd24 pacific blue, by BioLegend (2 mentions)
Facs 2 sorp cell sorter, by BD (2 mentions)
Ter119, by BD (2 mentions)
Cd11b pe cy5, by BioLegend (2 mentions)
Cd71 pe cy7, by BioLegend (2 mentions)
Cd41 bv605, by BioLegend (2 mentions)
Ter 119 bv421, by BioLegend (2 mentions)
Fcεriα af700, by BioLegend (2 mentions)
Ly 6g c fitc, by BD (2 mentions)
Cd3 pe, by BioLegend (2 mentions)
B220 pe cy7, by BioLegend (2 mentions)
Annexin 5 apc, by BioLegend (2 mentions)
Cd150 apc, by BioLegend (2 mentions)
8 protocols using cd117 apc cy7
1
Isolation and Flow Cytometry of Intestinal Stem Cells
2
Quantifying Mouse Limb Bud Cell Subsets
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For sample acquisition to determine the percentage of sSca+ and sSca– subpopulations in E10.5, E11.5, and E12.5 hindlimb cells, one million mouse limb bud cells were resuspended in 100 μL of FACS buffer containing a mixture of CD29-APC (1:100; cat. 102215), Sca-1-PE/Cy7 (1:300; cat. 108113), CD44-FITC (1:300; cat.103007), CD117-APC/Cy7 (1:100; cat 105825), and CD45-PerCP/Cy5 (1:500; cat. 103131), all from BioLegend (San Diego, CA, United States), and PE anti-mouse Flk1 (1:300, cat. no. 555308, BD Pharmigen, San Jose, CA, United States) antibodies. The cell suspension was incubated for 30 min on ice protected from light. Unlabeled cells and isotype antibodies were used as controls to exclude non-specific fluorescence. For cell sorting, between fifteen and twenty million mouse hindlimb bud cells were stained for each experiment. Sample acquisition was performed using a flow cytometer, Attune NXT (Life Technologies), and sorting was done using FACS Aria II (BD Biosciences, San Jose, CA, United States), with at least 20,000 events being collected. Data were analyzed using FlowJo Software version 10. Single cells were selected from the dot plot of side scatters height and area, and gates were determined by the FMO isotype control to scatter. Each acquisition and sorting experiments were run in triplicate and represented a pool of approximately 60 and 200 hindlimb buds, respectively.
3
Comprehensive Immune Cell Profiling
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2 x 106 cells were resuspended in 1 mg/mL beriglobin in PBA-E, incubated for 5 min and subsequently stained according to manufacturers recommendation with following antibodies in PBA-E in darkness at 4°C for 20 min. Set 1: CD45 V500 (clone HI30, BD Biosciences), CD15 BV605 (clone W6D3, BD Biosciences), CD14 FITC (clone M5E2, BD Biosciences), CD163 PE (clone GHI/61, BD Biosciences), CD32b PE/Cy7 (clone FUN-2, Biolegend), CD16 PerCP-Cy5.5 (clone 3G8, BD Biosciences), CD206 Alexa Fluor 700 (clone 15-2, Biolegend) and CD64 APC-H7 (clone 10.1, BD Biosciences); Set 2: CD45 V500 (clone HI30, BD Biosciences), FceRI BV605 (clone AER-37 (CRA1), BD Biosciences), CD141 FITC (clone JAA17, ThermoFisher (eBioscience)), CD303 PE/Cy7 (clone 201A, Biolegend), CD1c PerCP-Cy5.5 (clone F10/21A3, BD Biosciences), CD11c APC (clone N418, Biolegend), HLA-DR Alexa Fluor 700 (clone G46-6 (L243), BD Biosciences), CD117 APC/Cy7 (clone 104D2, Biolegend). Cells were washed with PBA-E and analysed on a FACS Canto-Plus flow cytometer (Becton Dickinson). Prior analysis, 1 µg/mL DAPI (4′,6-diamidino-2-phenylindole, Cell Signaling Technology) was added to each sample. Data were analysed with FlowJo V10.7 (BD).
4
Erythroid Lineage Differentiation Assay
5
Comprehensive Immune Cell Profiling
6
Erythroid Lineage Differentiation Assay
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Sorted cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) in the presence of 20% FCS supplemented with SCF (50 ng/mL), IL-3 (10 ng/mL), IL-6 (10 ng/mL), EPO (2 U/mL), TPO (50 ng/mL), IL-11 (50 ng/mL) and IL-5 (10 ng/mL) for 7 days. Cells were collected on days 2, 5 and 7, and labeled with the following cell surface markers for flow cytometric analysis:
TER119-BV421 (Clone TER-119, #116233 Biolegend)
CD71-PE Cy7 (Clone RI7217, #113812] Biolegend)
CD117-APC Cy7 (Clone 2B8, #105826 Biolegend)
FcεRIα-AF700 (Clone MAR-1,#134323 Biolegend)
CD41-BV605 (Clone MWReg30, #133921 Biolegend)
Cd11b-PE Cy5 (Clone M1/70, #101209 Biolegend)
Ly 6G/C-FITC (Clone RB6-8C5, #553126 BD Biosciences).
7
Comprehensive Immune Cell Profiling
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The following antibodies were used (all from BioLegend): CD3 (clone 17A2), Ter119 (TER-119), Gr-1 (RB6–8C5), B220 (RA3–6B2) conjugated to Pacific Blue, CD117 APC-Cy7 (2B8), Sca-1 PE-Cy7 (D7), Flk2 PE (A2F10), CD48 FITC (HM48–1), CD150 APC (TC15–12F12.2), B220 PE-Cy7 (RA3–6B2), Mac-1 PE-Cy5 (M1/70), CD3 PE (17A2), Ter119 APC-Cy7 (TER-119), CD45.2 FITC (104), CD45.1 APC-Cy7 (A20), CD16/32 PerCP-Cy5.5 (93), CD5 APC-Cy7 (53–7.3). CD19 PE (eBio1D3) and CD34 FITC (RAM34) were from eBioscience. Cells were labeled with SYTOX Blue viability stain (Thermo). Data were acquired on LSRII or LSR Fortessa instruments (BD Biosciences). APC-annexin V was from BioLegend.
8
Isolation and Flow Cytometry of Intestinal Stem Cells
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Following crypt isolation, the crypt suspensions were pelleted (100 g, 5 min, 4°C) and supernatant was discarded. Crypts were resuspended in TrypLE (GIBCO, no phenol red, 12604039) and dissociated into individual cells by warming crypts in water bath at 32°C for 60 s. Dissociated single cells were treated with the following antibody cocktail for flow cytometry analysis: CD45-PE (eBioscience, 12-0451-83), CD31-PE (Biolegend, 102514), Ter-119PE (Biolegend, 116208), CD24-Pacific Blue (Biolegend, 101820), CD117-APC/Cy7 (Biolegend, 105826), and EpCAM-APC (eBioscience, 17-5791-82). 7AAD (Invitrogen, A1310) was used a viability dye to exclude dead cells from the analysis. ISCs were isolated as Lrg5-eGFPhiEpCAM+CD24low/−CD31−Ter119−CD45−7AAD−. eGFPlow progenitors were isolated as Lrg5-eGFPlowEpCAM+CD24low/−CD31−Ter119−CD45−7AAD−. Cells were sorted using a BD FACS II SORP cell sorter.
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