Trypsinized cells were filtered through a 40-μm sterile cell strainer (Fisher) and washed with DMEM supplemented with FBS. Alternatively, tumors were harvested from mice, dissociated using the GentleMACS dissociator kit (Miltenyi Biotec), incubated in ACK red blood cell lysis buffer (Invitrogen), and filtered through a 40-μm sterile cell strainer. ALDH activity was quantified using the Aldefluor kit (Stem Cell Tech.) Briefly, single-cell suspensions of 106 cells were incubated in activated Aldefluor substrate (BAAA), or equivalent volume of Aldefluor inhibitor diethyl aminobenzaldehyde (DEAB). CD44 was probed with anti-CD44 (APC #559942, BD Pharmingen or V450 #561292, BD Horizon). 5T4 was probed using MEDI0641 (MedImmune) for 1 hour on ice, followed by labeling with anti-human Alexa Fluor 594 (#A11014; Invitrogen) for 30 minutes at 4°C. Human cells were distinguished by positive anti-HLA-ABC staining (PE #560168; BD Pharmingen), and 7-aminoactinomycin (7AAD #00-6993-50, eBioscience) was used to isolate viable cells. For cell cycle analysis, filtered cells were fixed in 70% ethanol for 1 hour, followed by incubation with propidium iodide (PI #P4864, Sigma) for 30 minutes at 4°C.
Apc 559942
Manufactured by BD
Sourced in United States
The APC #559942 is a laboratory instrument used for the detection and analysis of cellular antigens. It is designed to provide accurate and reliable results in flow cytometry applications. The core function of this product is to enable the detection and quantification of specific cell surface proteins or intracellular markers.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Ack red blood cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
40 μm sterile cell strainer, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Facsdiva, by BD (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Flowlogic software, by Miltenyi Biotec (1 mentions)
70 μm cell strainer, by BD (1 mentions)
2 protocols using apc 559942
1
Isolation and Characterization of Tumor Cells
2
Isolation of Tumor Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We minced each sample into 1-mm 3 pieces with a scalpel and incubated them in Accutase (LINUS) for 1 h at 37 °C. We filtered the cell suspensions with a 70 μm cell strainer (Falcon, NY, USA) and assessed cell viability with Trypan Blue (Gibco, MA, USA). When the percentage of dead cells exceeded 30%, we performed a Ficoll-Paque density gradient centrifugation to discard dead cells and debris before sorting. We washed the cell pellets twice and re-suspended them in ice-cold phosphate-buffered saline (PBS). Then, we incubated them with antibodies against the epithelial cell adhesion molecule (EpCAM) (EBA1, FITC, 347197) and stem cell markers CD44 (APC, 559942), CD166 (PE, 559263), and LGR5 (BV 421, 562925) (all from BD Biosciences, NJ, USA). DRAQ5 (Thermo Fisher Scientific, MA, USA) and 7AAD (Invitrogen, MA, USA) dyes were additionally used to select nucleated cells and exclude non-viable ones. Individual tumor cells were gated and sorted into stem cell (EpCAM+/CD44+/CD166+/Lgr5-; EpCAM-/CD44-/CD166-/Lgr5+) and non-stem cell (EpCAM+/CD44-/CD166-/Lgr5-) phenotypes (Fig. S1). We carried out cell sorting using a FACSAria III (BD Biosciences, NJ, USA) flow cytometer and analyzed the resulting data using FACSDiva (BD Biosciences, NJ, USA) and FlowLogic software (Miltenyi Biotec, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!