Protocols were adopted from our previous study12 (link). Prostate tissues were processed as described above for bPSC isolation and isolated cells were stained with the following fluorescence conjugated antibodies (Biolegend): CD45-PerCP (#103130), Sca-1-APC (#122512) and CD49f-PE (#313612) before fixation with 10% neutral buffered formalin. To detect AR protein, fixed samples were stained with unconjugated rabbit anti-AR (#ab52615, Abcam, 1:50) followed by secondary stain with Alexa Fluor 488 (Invitrogen, 1:500). Prostate cells isolated from lineage tracing mice were stained with the following fluorescence conjugated antibodies (Biolegend): EpCAM-PE/Cyanine7 (#18215) and CD49f-BV421(#313623). Flow cytometry analyses were performed using BD LSRFortessa.
Sca1 apc
Manufactured by BioLegend
Sourced in United States
Sca1-APC is a fluorochrome-conjugated antibody that recognizes the Sca1 (Stem Cell Antigen-1) cell surface marker. Sca1 is expressed on various stem and progenitor cell populations in mice. The APC (Allophycocyanin) fluorochrome allows for flow cytometric detection and analysis of Sca1-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 fitc, by BioLegend (5 mentions)
Cd45 apc, by BioLegend (4 mentions)
Cd45.1 apc, by BioLegend (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Cd49f pe, by BioLegend (3 mentions)
C kit apccy7, by BioLegend (3 mentions)
Cd45 pe, by BioLegend (3 mentions)
Facsaria 2, by BD (3 mentions)
Collagenase 1, by Merck Group (3 mentions)
Facsaria, by BD (3 mentions)
Cd31 fitc, by BioLegend (3 mentions)
Cd49f bv421, by BioLegend (2 mentions)
Cd29 apc, by BioLegend (2 mentions)
Cd3e pe, by BioLegend (2 mentions)
Cd14 fitc, by BioLegend (2 mentions)
Cd15 pacific blue, by BioLegend (2 mentions)
Glycophorin pecy5, by BioLegend (2 mentions)
Clone hir2, by BioLegend (2 mentions)
Mac 1 cd11b pecy7, by BioLegend (2 mentions)
Gr 1 pe, by BioLegend (2 mentions)
25 protocols using sca1 apc
1
Prostate Cell Isolation and Characterization
2
Multicolor Immunophenotyping of Modified MSCs
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Sterile cover glasses were placed on a 6 cm plate and 7.5 × 105 modified MSC suspended in IMDM culture medium with FBS were incubated under standard culture conditions (37 °C, 5% CO2) for 24 h. Cover glasses were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained with antibodies conjugated with fluorochrome: CD29-APC, CD90-FITC, Sca-1-APC, CD45-APC (BioLegend, USA). Microscopic observations were performed using an LSM 710 Zeiss confocal microscope (Carl Zeiss Microscopy GmGB, Gottingen, Germany).
3
Murine Hematopoietic Cell Isolation
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Mice were bled into 150 µL of Alsever’s solution. Samples were then treated with 10 mL ACK buffer for 2 min and centrifuged at 1600 RPM for 5 min. The supernatant was removed, and pellets were washed in 5 mL sample media (SM, PBS with 2 mM EDTA and 2% FCS). Antibodies of Biolegend (San Diego, CA, USA): CD45.1 APC, CD45.2 pacific blue, Mac1 PE-Cy7, B220 APC-Cy7, CD3e PE, Lineage-PacificBlue (Including- anti Ter119, Mac1, Gr1, CD3e, CD4, CD8 and B220), c-Kit Alexa780, Sca1-APC, CD150-PEcy7, CD48- Percp cy5.5, CD9-APC, CD34-PE, CD84-PE. Cells were stained on ice for 1 hr and washed. Flow cytometry analysis of the reporter expression frequencies (ZsGreen+) and the surface markers’ expression were performed on the Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA). Fluorescence activated cell sorting (FACS) data analysis was performed using Kaluza v1.2 (Beckman Coulter). Sorting was performed on FACSAria III (BD), as previously reported [9 (link)].
4
Hematopoietic Progenitor Cell Analysis
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Cells were suspended in Ca++ Mg++-free phosphate-buffered saline (PBS) containing 2% (vol/vol) FBS (Globalstem). Human hematopoietic progenitors cells were identified by labeling with CD45-PE (1:50; clone HI30) and or CD34-APC (1:50; clone 561) while mature phenotypes were analyzed using CD15-Pacific Blue (monocytes and granulocytes) (1:50; clone W6D3/HI98), CD14-FITC (monocytes and macrophages)(clone 1:50; M5E2); CD11b (1:50; ICRF44) (macrophages) and Glycophorin-PECy5 (red blood cell membrane), and erythroid precursors (1:50; Clone HIR2) (all from Biolegend). Murine hematopoietic cells were identified using the following antibodies: Mac-1/CD11b-PECy7 (1:200; clone M1/70), Gr-1-PE (1:200; clone 8C5), B220-PECy5 (1:100; clone RA3-6B2), Sca1-APC (1:100; clone E13-161-7), c-Kit-APCCy7 (1:100; clone 2B8): CD45.1-APC (1:100; clone A20) and CD45.2-FITC (1:100; clone 104) (all from Biolegends). All antibodies were incubated at the concentration suggested by the manufacturer for 30 minutes on ice. Non-specific signals and dead cells were excluded by appropriate fluorochrome-conjugated isotype and propidium iodide staining, respectively. Cell fluorescence was analyzed with the FACSAria II (Becton Dickinson).
5
Phenotypic Characterization of MSC-Derived EVs
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Surface markers on MSC-EVs were analyzed using Apogee A50-Micro flow cytometer (Apogee Flow Systems, UK). The following fluorochrome-conjugated antibodies against murine antigens were used according to the manufacturer’s protocols: CD29-APC (clone: HMβ1-1, Biolegend), CD44-APC (clone: IM7, Biolegend), CD81-APC (clone: Eat2, BD Bioscence), CD90-APC (clone: 30-H12, Biolegend), CD309-APC (clone: Avas12, Biolegend) and Sca-1-APC (clone: E13-161.7, Biolegend) as well as the following isotype controls: Armenian hamster IgG-APC (clone: HTK888, Biolegend), Rat IgG2a, κ-APC (clone: RTK2758, Biolegend) and rat IgG2b, κ-APC (clone: RTK4530, Biolegend). MSC-EVs were co-stained with SYTO RNA Select dye (ThermoFisher Scientific), which binds RNA molecules. Staining was conducted for 30 min in the dark at 4 °C. The obtained results were analyzed using Apogee Histogram software (Apogee Flow Systems). In order to confirm the presence of the indicated antigens on MSC-EVs, an ImageStream X Mark II imaging cytometer (Merck) was additionally used.
6
Magnetic Isolation of Neutrophils and LSK+ Cells
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For magnetic isolation of blood neutrophils, red blood cells were lysed as above, and single cell suspensions were washed in selection media. Cells were then incubated with Fc block for 10 min on ice as noted above. Neutrophils were isolated using the MojoSort Mouse Ly6G Selection Kit (Biolegend, Cat# 480,058) according to manufacturer’s instructions. For magnetic isolation of femur bone marrow LSK + cells, single cell suspensions were blocked and stained with biotinylated-lineage cocktail (Biolegend, Cat# 133,307) and stem/progenitor cells were negatively selected using streptavidin-conjugated nanobeads (Biolegend, Cat# 480,015). Lineage-negative cells were then stained with c-Kit-APC (Biolegend, Cat# 105,811) for positive selection with anti-APC-conjugated nanobeads (Biolegend, Cat# 480,071). Finally, Lin-cKit + bone marrow cells were stained with Sca1-APC (Biolegend, Cat# 108,111) for positive selection with anti-APC-conjugated nanobeads. The resulting neutrophil and LSK + cell populations were stored in Qiazol lysis reagent (Qiagen, Cat# 79,306) at -80 °C.
7
Multiparametric Immunophenotyping of Hematopoietic Cells
8
Isolation and Characterization of Muscle Stem Cells
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Gastrocnemius muscles were minced and digested in collagenase B/dispase II for 1 hr with trituration every 15 min. Digestions were neutralized with FBS and pelleted at 350 X g. Samples used to analyze for SCs were stained with CD31‐APC, CD45‐APC, Sca1‐APC, and Vcam‐1‐biotin antibodies (BioLegend, San Diego, CA). After a brief wash, streptavidin‐PE‐Cy7 conjugated secondary antibody (BioLegend, San Diego, CA), together with PI (selection for dead cells) and calcein violet (selection for live cells) (BioLegend, San Diego, CA), was added prior to analysis. Samples were run through a FACSAria I (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR).
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Gastrocnemius muscles were minced and digested in collagenase B/dispase II for 1 hr with trituration every 15 min. Digestions were neutralized with FBS and pelleted at 350 X g. Samples used to analyze for SCs were stained with CD31‐APC, CD45‐APC, Sca1‐APC, and Vcam‐1‐biotin antibodies (BioLegend, San Diego, CA). After a brief wash, streptavidin‐PE‐Cy7 conjugated secondary antibody (BioLegend, San Diego, CA), together with PI (selection for dead cells) and calcein violet (selection for live cells) (BioLegend, San Diego, CA), was added prior to analysis. Samples were run through a FACSAria I (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR).
9
Murine Skeletal Muscle Stem Cell Isolation
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Both TA muscles from 1 mouse were pooled, finely minced, and digested for 60 minutes at 37°C in DMEM with 0.2% (~5500 U/mL) collagenase type II and 2.5 U/mL dispase II. The resulting digest solution was filtered through a 70 μm cell strainer and then centrifuged at 350g for 5 minutes. Cells were resuspended and incubated for 30 minutes on ice with primary antibodies, including Sca-1-APC (BioLegend clone D7; 108112), CD45-AF488 (BioLegend clone 30-F11; 103121), CD11b-APC (BioLegend clone M1/70; 101212), Ter119-APC (BioLegend clone TER-119; 116212), CD29/β1-integrin-PE (BioLegend clone HMb-1; 102208), and CD184/CXCR-4-BIOTIN (BD Biosciences lot 6336587; 551968). Cells were then washed, centrifuged, and resuspended in PECy7-STREPTAVIDIN secondary antibody (eBioscience, Thermo Fisher Scientific, lot 4290713; 25-4317-82). The stained cells were filtered through a 35 μm cell strainer, propidium iodide viability dye was added (1 μg) (Thermo Fisher Scientific), and FACS was performed using a BD FACSAria III Cell Sorter (BD Biosciences). MuSCs, classified here as APC/FITC (Sca-1, CD11b, Ter119, CD45) double-negative and PE/PECy7 (β1-integrin/CXCR-4) double-positive cells, were sorted into TRIzol reagent (Thermo Fisher Scientific) and stored at –80°C.
10
Flow Cytometric Analysis of Hematopoietic Stem Cells
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Peripheral blood samples were collected, and white blood cells were obtained after red blood cell (RBC) lysis. Cells were suspended in staining buffer (BioLegend); treated with 0.5 µL of Trustain (BioLegend), followed by fluorescent-conjugated antibodies, Lineage cocktail-FITC, Sca-1-APC, and c-Kit-PE (BioLegend); and were incubated for 45 min at 4°C. Cells were resuspended in PBS, and 7-AAD (BD Pharmingen) was added to detect dead cells. No-stain control samples were prepared in the same way with isotype controls of respective antibodies. Flow cytometry was performed by using the Accuri-C6 System, and data were analyzed according to the gating strategy shown in Supplementary Fig. 1 to enumerate LSK cells (26 (link)).
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