BM hematopoietic cells were isolated from the femurs and tibias by flushing and the depletion of red blood cells by ACK Lysing Buffer (Thermo Fisher Scientific). The cells were stained with the appropriate dilution of fluorochrome- and/or biotin-conjugated antibodies and 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI) for dead cell exclusion and analysis using a FACSVerse flow cytometer and FASCAria (BD Biosciences). The following antibodies (all BioLegend) were used: Lineage Cocktail [CD5(53-7.3)-biotin, TER-119(TER-119)-biotin, CD11b(M1/70)-biotin, Gr-1(RB6-8C5)-biotin and B220(RA3-6B2)-biotin, 1:1000 each] with streptavidin BV605 (1:500), c-Kit(2B8)-PE-Cy7(1:500), Sca-1(D7)-APC(1:500) and CD27(LG.3A10)-BV421 (1:500) for mouse CML LSC analysis; CD34(561)-APC-Cy7(1:500), CD38(HB-7)-PE-Cy7(1:500), CD90(5E10)-biotin(1:500) with streptavidin BV605(1:500), CD45RA(HI100)-FITC(1:500), CD45(2D1)-FITC(1:250) or -BV421(1:250), Lineage Cocktail [CD3(OKT3), CD14(M5E2), CD16(3G8), CD19(HIB19), CD20(2H7), CD56(HCD56), 1:100]-BV510 for human CML LSC analysis; and CD274(10F.9G2 and 29E.2A3)-PE(1:500) for the detection of mouse and human PD-L1, respectively. The detection of annexin V-positive cells for apoptosis analysis was performed using either Annexin V-PE or APC.
Lineage cocktail
Manufactured by BioLegend
Sourced in United States
The Lineage Cocktail is a set of reagents designed to detect specific cell surface markers using flow cytometry. The cocktail contains a combination of fluorochrome-conjugated antibodies that allow for the identification and enumeration of various cell types within a sample. The core function of the Lineage Cocktail is to provide a standardized and efficient means of characterizing complex cellular populations.
Automatically generated - may contain errors
Lab products found in correlation
Sca 1, by BioLegend (2 mentions)
Primaryanti p stat5, by BD (2 mentions)
Anti p jak2 antibody, by Cell Signaling Technology (2 mentions)
Fcgamma receptor, by BD (2 mentions)
Phosflow perm buffer 3, by BD (2 mentions)
Cd11b, by BD (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (2 mentions)
Cd45 clone 104, by BioLegend (1 mentions)
Cd45.1 clone a20, by BD (1 mentions)
Cd45.2 clone 104, by BioLegend (1 mentions)
Facsaria 3, by BD (1 mentions)
Tyro3, by R&D Systems (1 mentions)
Mhcii, by BD (1 mentions)
Mertk, by Thermo Fisher Scientific (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Hla dr, by BD (1 mentions)
F4 80, by BD (1 mentions)
Cd135, by BD (1 mentions)
Sca 1 apc, by Thermo Fisher Scientific (1 mentions)
7 protocols using lineage cocktail
1
Isolation and Characterization of CML Leukemic Stem Cells
2
Multiparametric Flow Cytometry for Hematopoietic Stem Cells
3
Immunophenotyping of Murine HSCs and Progenitors
4
Immunophenotyping of Murine HSCs and Progenitors
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Human TF-1 cells were fixed with 1% formaldehyde after
stimulation (with 10ng/ml rhFL, T or FL:T IL-3), permeabilized using BD Phosflow
Perm Buffer III (#558050) and stained in BD staining buffer with primary
anti-P-Stat5 (BD) and anti-P-Jak2 antibodies (Cell Signaling) and secondary
antibody (Cell signaling). Mouse bone marrow was washed in PBS and
2×106 cells were stained with the following phenotyping
antibodies to determine HSC and progenitor populations29 (link) prior to fixation [Lineage
cocktail (Biolegend,), CD34, SCA, KIT, FLT3, Fcgamma receptor (BD)]. All
samples were run on BD machines (LSR 4 or Fortessa) and analyzed using FlowJo
Software.
stimulation (with 10ng/ml rhFL, T or FL:T IL-3), permeabilized using BD Phosflow
Perm Buffer III (#558050) and stained in BD staining buffer with primary
anti-P-Stat5 (BD) and anti-P-Jak2 antibodies (Cell Signaling) and secondary
antibody (Cell signaling). Mouse bone marrow was washed in PBS and
2×106 cells were stained with the following phenotyping
antibodies to determine HSC and progenitor populations29 (link) prior to fixation [Lineage
cocktail (Biolegend,), CD34, SCA, KIT, FLT3, Fcgamma receptor (BD)]. All
samples were run on BD machines (LSR 4 or Fortessa) and analyzed using FlowJo
Software.
5
Multiparametric Analysis of Myeloid Cells
6
Isolation and Characterization of Murine Hematopoietic Stem and Progenitor Cells
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Femurs were obtained from mice immediately after cervical dislocation. Femurs were punctured at both ends and centrifuged to pellet marrow cells in PBS + 10% FBS. Bone marrow cells were incubated with red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) for 5 min at room temperature to remove erythrocytes. Remaining cells were lineage depleted with an EasySepMouse Haematopoietic Progenitor Isolation Kit (Stem Cell Technologies #19856). Lineage depleted cells were stained in PBS + 5% FBS + brilliant stain buffer (BD #563794) with the following antibodies: Lineage cocktail (BV421, Biolegend #133311), CD117 (PEcy7, eBioscience #25117182), Sca1 (APC, eBioscience #17598183), CD34 (FITC, BD #553733), CD135 (PE, BD #553842), CD48 (APCcy7, Biolegend #103432), CD150 (BV650, Biolegend #115931), CD16/32 (APCcy7, Biolegend #101328) and CD127 (BV711, Biolegend #135035). Cells were washed thoroughly and analyzed on a BD LSR Fortessa FACS analyser. The gating strategy is described in Supplementary Fig. 8 .
For colony-forming assays, lineage negative cells obtained from the bone marrow were counted and 10,000 cells were plated in methylcellulose semi-solid medium (MethocultGF M3434, Stem Cell Technologies). Cells were allowed to grow for 7 days and counted using an upright microscope to score colony type and number as per the manufacturers’ instructions.
For colony-forming assays, lineage negative cells obtained from the bone marrow were counted and 10,000 cells were plated in methylcellulose semi-solid medium (MethocultGF M3434, Stem Cell Technologies). Cells were allowed to grow for 7 days and counted using an upright microscope to score colony type and number as per the manufacturers’ instructions.
7
Murine Hematopoietic Lineage Analysis
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Bone marrow cells were flushed from the femurs and tibias of mice with PBS containing 2% FBS and lysed with lysing buffer (catalogue number: R1010, Solarbio, China) for red blood cell deletion. Bone marrow cells were suspended in PBS and stained by antibodies for 30 min. For Detection lineage-specific markers: Erythroid: Ter119 (FITC, Biolegend, catalogue number: 116206) and CD71 (PE, eBioscience, catalogue number:12–0711-82), Granular line: CD11b (FITC, BD, catalogue number:557396) and Gr-1 (PE, BD, catalogue number: 553126), Megakaryocyte: CD41 (FITC, BD, catalogue number:553848) and C-kit (PE, BD, catalogue number:553355), LSK: lineage cocktail (FITC), sca-1 (APC, Biolegend, catalogue number:108112) and C-kit (PE, BD, catalogue number:553355). Cell apoptosis analysis was performed using Annexin V staining kit according to the instructions of the manufacturer (BD).
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