Lung cell suspensions were prepared using an elastase digestion and stained for fluorescence-activated cell sorting (FACS), as previously described (20 (link)). Briefly, cells were resuspended in Hanks' balanced saline solution buffer supplemented with 2% fetal bovine serum, 0.1 mM EDTA, 10 mM HEPES, 100 IU/ml penicillin and 100 µg/ml streptomycin (HBSS+). Cells were then stained with the primary antibodies on ice for 45 min. The following antibodies were employed: EpCAM-PE-Cy7 (25-5791-80, 1:100), CD31-Biotin (13-0311-81, 1:40), CD34-Biotin (13-0341-81, 1:10), CD45-Biotin (13-0451-81, 1:100), Sca-1-APC (17-5981-81, 1:100), and CD24-PE (12-0242-81, 1:20) (all from eBioscience, Inc., San Diego, CA, USA). Cells were subsequently stained with the secondary antibody on ice for 40 min using streptavidin-APC-Cy7 (47-4317-82, 1:100; eBioscience, Inc.). Dead cells were identified using 7-aminoactinomycin D staining (BD Biosciences, Franlkin Lakes, NJ, USA).
Streptavidin apc cy7
Manufactured by Thermo Fisher Scientific
Sourced in United States
Streptavidin-APC-Cy7 is a fluorescent conjugate that combines the high-affinity biotin-binding properties of streptavidin with the APC-Cy7 fluorochrome. It is commonly used in flow cytometry and other bioanalytical applications that require the detection and quantification of biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Cd24 pe, by Thermo Fisher Scientific (2 mentions)
Cd31 biotin, by Thermo Fisher Scientific (2 mentions)
Cd34 biotin, by Thermo Fisher Scientific (2 mentions)
Cd45 biotin, by Thermo Fisher Scientific (2 mentions)
Sca 1 apc, by Thermo Fisher Scientific (2 mentions)
Streptavidin fitc, by BioLegend (2 mentions)
Streptavidin pe, by BioLegend (2 mentions)
7 amino actinomycin d staining, by BD (1 mentions)
Epcam pe cy7, by Thermo Fisher Scientific (1 mentions)
Mac 1 m1 70, by Thermo Fisher Scientific (1 mentions)
Cluster tube, by Corning (1 mentions)
7 aminoactinomycin d 7 aad, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Epcam pe cy7, by BioLegend (1 mentions)
5 protocols using streptavidin apc cy7
1
Lung Cell Isolation and FACS Analysis
2
Multicolor Flow Cytometry Panel
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Purified antibodies specific for CD3ε (145–2C11), CD4 (L3T4), CD5 (53–7.3), CD8 (53–6.7), B220 (RA3–6B2), Gr-1 (RB6–8C5), Ter119 (Ter-119), CD16/32 (93) and Mac-1 (M1/70) were obtained from eBioscience (San Diego, CA, USA). Fluorochrome-conjugated antibodies specific for CD11c (N418), CD11b (M1/70), and streptavidin-APC-Cy7 were obtained from eBioscience. Fluorochrome-conjugated antibodies specific for CD3 (145–2C11), CD8 (53–6.7), CD19 (MB19–1), Gr-1 (RB6–8C5), Ter119 (Ter-119), B220 (RA3–6B2), Ly6C (AL-21), Ly6G (1A8), MHC-II (AF6–120.1), F4/80 (C1: A3–1), c-kit (2B8), Sca1 (E13–161.7), Flt3 (A2F10), CD150 (TC15–12F12.2), 4–1BBL (TKS-1), streptavidin-PE-Cy7, streptavidin-PE and streptavidin-FITC were obtained from Biolegend (San Gabriel, CA, USA). Isotype control antibodies including Rat IgG2a (R35–95), Rat IgG2b (RTK4530), Rat IgG2b (eB149/10H5), Mouse IgG2a (eBM2a) and Hamster IgG (eBio299Arm) were obtained from eBioscience. All antibodies were titrated prior to use to determine concentration giving minimum saturation binding.
3
Multicolor Flow Cytometry Phenotyping
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Cells were sedimented into the wells of a flexible 96-well polystyrene microtitre plate (Corning, New York, USA). For FcR blocking, cells were resuspended in 25 μl anti-CD16/32 followed by incubation on ice for 15 min. After washing, cells were resuspended in a primary antibody cocktail (APC-conjugated CD11c (N418), PE-Cy7-conjugated CD11b (M1/70), PE-conjugated CD8α (53-6.7), biotin- or FITC-conjugated MHC-II (AF6-120.1) all from eBioscience) and incubated on ice for 25 min. before washing. Where necessary, secondary labelled conjugate (streptavidin-APC-Cy7 from eBioscience) was added and cells were incubated for 25 min. on ice, washed and transferred to a cluster tube (Corning) for analysis performed with a flow cytometer (Becton Dickinson LSRII, San Jose, CA, USA). Prior to analysis, propidium iodide (PI: 1000 μg/ml) was added for dead cell discrimination.
4
Multiparametric Flow Cytometry Panel
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Fluorochrome-conjugated antibodies specific for CD4 (GK1.5), Thy1.2 (30-H12), CD69 (H12F3), B220 (RA3-6B2), MHC-II (AF6–120.1), F4/80 (C1: A3–1), CD8α (53–6.7), Sirp-α (P84), 4-1BBL (TKS-1), streptavidin-PE-Cy7, streptavidin-PE and streptavidin-FITC were obtained from Biolegend. Fluorochrome-conjugated antibodies specific for CD11c (N418), CD11b (M1/70), CD115 (AFS98) and streptavidin-APC-Cy7 were obtained from eBiosciences (San Diego, CA, USA) or Biolegend (San Gabriel, CA, USA). Isotype control antibodies including Rat IgG2a-FITC (R35–95), Rat IgG2b-PE (RTK4530), Rat IgG2b-PE-Cy7 (eB149/10H5), Mouse IgG2a-biotin (eBM2a) and Hamster IgG-APC (eBio299Arm) were obtained from eBioscience.
5
Isolation and Characterization of Mouse Lung Cell Types
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A single-cell suspension was prepared by digestion of lung tissues from 6–10-week-old C57BL/6 mice with elastase and DNase I. Freshly isolated cells were suspended in Hanks balanced saline
solution (Solarbio, Beijing, China) with 2% fetal bovine serum (FBS, Gibco), 0.01% Penicillin- streptomycin (Gibco, USA) and 10 mM HEPES (Sigma). The primary antibodies used to stain cells
included CD24-PE (1:25, eBioscience, San Diego, CA, USA), EpCAM-PE-Cy7 (1:50, Biolegend, San Diego, CA, USA), CD31-biotin (1:50, eBioscience), CD45-biotin (1:100, eBioscience), CD34-biotin
(1:16, eBioscience), and Sca-1-APC (1:100, eBioscience). Then, the secondary antibody Streptavidin-APC-Cy7 (1:100, eBioscience) was used, followed by the addition of 7-amino-actinomycin D
(7-AAD, 1:20, eBioscience) to discriminate the dead cells. Mouse club and AT2 cells were sorted based on their surface expression pattern,
CD31−CD34−CD45−EpCAM+CD24+Sca-1+ and
CD31−CD34−CD45−EpCAM+CD24−Sca-1−, respectively.
solution (Solarbio, Beijing, China) with 2% fetal bovine serum (FBS, Gibco), 0.01% Penicillin- streptomycin (Gibco, USA) and 10 mM HEPES (Sigma). The primary antibodies used to stain cells
included CD24-PE (1:25, eBioscience, San Diego, CA, USA), EpCAM-PE-Cy7 (1:50, Biolegend, San Diego, CA, USA), CD31-biotin (1:50, eBioscience), CD45-biotin (1:100, eBioscience), CD34-biotin
(1:16, eBioscience), and Sca-1-APC (1:100, eBioscience). Then, the secondary antibody Streptavidin-APC-Cy7 (1:100, eBioscience) was used, followed by the addition of 7-amino-actinomycin D
(7-AAD, 1:20, eBioscience) to discriminate the dead cells. Mouse club and AT2 cells were sorted based on their surface expression pattern,
CD31−CD34−CD45−EpCAM+CD24+Sca-1+ and
CD31−CD34−CD45−EpCAM+CD24−Sca-1−, respectively.
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