Tumors were excised on day 20 post-inoculation and processed on GentleMACS Octo Dissociators using Mouse Tumor Dissociation Kits (both from Miltenyi Biotec, San Diego, CA, USA). Resulting single-cell suspensions were stained with LIVE/DEAD-Near IR viability dye (Thermo Fisher, Carlsbad, CA, USA) and Brilliant Violet 650-conjugated anti-mouse CD45 antibody (BioLegend, San Diego, CA, USA) according to manufacturer’s recommendations. Data were acquired on a Novocyte flow cytometer and analyzed using NovoExpress software (ACEA Biosciences, San Diego, CA, USA). GFP-expressing 3T3HAS3 fibroblasts were gated as live CD45- GFP+ cells and quantified as a percentage of the CD45- population (non-hematopoietic cells). For analyses of CD44 expression and HA binding of MDA-MB-468 shCD44 855 cells, cultured cells were dissociated with Accutase (BD Biosciences, San Jose, CA, USA) and stained with anti-mouse/human CD44 antibody IM7-phycoerythrin (eBioscience, San Diego, CA, USA) or sequential incubation with biotinylated HA [57 (link)] and streptavidin–phycoerythrin (SA–PE, R&D Systems, Minneapolis, MN, USA). CD44 expression and cell-associated HA levels in 3T3HAS3 CD44 KO cells were evaluated with the Alexa Fluor 488 anti-mouse/human CD44 antibody IM7 (BioLegend, San Diego, CA, USA) or sequential incubation with HTI-601 and SA–PE.
Live dead near ir viability dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LIVE/DEAD-Near IR viability dye is a fluorescent stain used to assess cell viability. It allows the discrimination between live and dead cells based on their membrane integrity. The dye emits fluorescence in the near-infrared region of the spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
Software, by FlowJo (2 mentions)
Ficoll paque premium, by GE Healthcare (2 mentions)
Ls column, by Miltenyi Biotec (2 mentions)
Anti pe microbeads, by Miltenyi Biotec (2 mentions)
Sh800 cell sorter, by Sony (2 mentions)
Mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Accutase, by BD (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Novoexpress software, by Agilent Technologies (1 mentions)
Sa pe, by BioLegend (1 mentions)
Cd25 bc96, by BioLegend (1 mentions)
Ctla 4 l3d10, by BioLegend (1 mentions)
Cd4 rpa t4, by BioLegend (1 mentions)
Cd69 fn50, by BioLegend (1 mentions)
Facs symphony flow cytometer, by BD (1 mentions)
6 protocols using live dead near ir viability dye
1
Quantification of Tumor-Infiltrating Fibroblasts
2
Activation Kinetics of CD4+ T Cells
3
Anti-CD3 Stimulated Calcium Flux
4
Isolation and Characterization of Porcine Thymic NKT Cells
5
Isolation and Characterization of Porcine Thymic NKT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 1 g of thymus tissue was collected from one male and
one female mix-breed pig at 22 weeks of age that were slaughtered at the
University of Florida’s Animal Sciences Department. Single cell
suspensions were generated using glass homogenizers, after which mononuclear
cells were isolated using Ficoll-Paque PREMIUM (GE Healthcare). Some cells
were set aside for scRNA-seq of total thymocytes. The remaining cells were
stained with Live/Dead Near-IR viability dye (Invitrogen) and a
phycoerythrin (PE)-conjugated alpha-galactosylceramide (aGC) analog
PBS57-loaded mouse CD1d (mCD1d) tetramer (National Institutes of Health
Tetramer Core Facility). Cell suspensions were subsequently incubated with
magnetically labeled anti-PE MicroBeads (Miltenyi Biotec), washed, and
loaded onto an LS column (Miltenyi Biotec). After washing, the labeled cells
were eluted and loaded onto an MS column for further enrichment. An equal
number of cells from both pigs were combined and sorted for live mCD1d
tetramer positive cells using a Sony SH800 Cell Sorter (Sony Biotechnology).
Aliquots from the sorted cells were co-stained with allophycocyanin
(APC)-conjugated PBS57-loaded or unloaded mCD1d tetramers to validate their
specificity for the CD1d tetramer-PBS57 antigen complex by FACS. The
thymocyte samples were stained with Live/Dead Near-IR viability dye and FACS
sorted for live cells.
one female mix-breed pig at 22 weeks of age that were slaughtered at the
University of Florida’s Animal Sciences Department. Single cell
suspensions were generated using glass homogenizers, after which mononuclear
cells were isolated using Ficoll-Paque PREMIUM (GE Healthcare). Some cells
were set aside for scRNA-seq of total thymocytes. The remaining cells were
stained with Live/Dead Near-IR viability dye (Invitrogen) and a
phycoerythrin (PE)-conjugated alpha-galactosylceramide (aGC) analog
PBS57-loaded mouse CD1d (mCD1d) tetramer (National Institutes of Health
Tetramer Core Facility). Cell suspensions were subsequently incubated with
magnetically labeled anti-PE MicroBeads (Miltenyi Biotec), washed, and
loaded onto an LS column (Miltenyi Biotec). After washing, the labeled cells
were eluted and loaded onto an MS column for further enrichment. An equal
number of cells from both pigs were combined and sorted for live mCD1d
tetramer positive cells using a Sony SH800 Cell Sorter (Sony Biotechnology).
Aliquots from the sorted cells were co-stained with allophycocyanin
(APC)-conjugated PBS57-loaded or unloaded mCD1d tetramers to validate their
specificity for the CD1d tetramer-PBS57 antigen complex by FACS. The
thymocyte samples were stained with Live/Dead Near-IR viability dye and FACS
sorted for live cells.
6
SIV Env Antibody Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CEM.NKR-CCR5-sLTR-Luc cells were infected with vif-deleted SIVmac239, with or without Env mutations and pseudotyped with VSV G by spinoculation at 1200 g for 1 hour in the presence of 40 μg/ml Polybrene. Antibody binding to Env was evaluated by staining the cells 3 days post-infection. The cells were treated with Live/Dead NEAR IR viability dye (Invitrogen), washed in staining buffer (PBS with 1% FBS), and then incubated on ice for 30 minutes with 10 μg/ml of Env-specific antibody or a 1:32 dilution of SIV+ plasma pool from chronically infected rhesus macaques used in other studies. A dengue virus-specific antibody (DEN3) was used as a negative control. An AF647-conjugated goat anti-human F(ab′)2 (Jackson Immunoresearch) and PE-Cy7-conjugated anti-CD4 (Clone OKT4, Biolegend) were used for subsequent staining on ice. For intracellular p27 staining, cells were fixed in PBS with 2% paraformaldehyde, washed in staining buffer, and stained with FITC-conjugated anti-SIV Gag antibody (clone 552F12) in Perm/Fix Medium B (Invitrogen). After washing, the cells were fixed in PBS with 2% paraformaldehyde and analyzed using a BD FACS Symphony flow cytometer. Antibody binding to Env was assessed by Env staining on the surface of SIV-infected (CD4lowGag+) cells.
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!