Cryopreserved PBMC and/or single cell tumor samples were thawed and Treg subsets were assessed by flow cytometry as described before [19 (link)]. Antibodies and kits used were V500-labeled CD3 (clone UCHT1, BD Biosciences), AF700- or PE-Cy7-labeled CD4 (clone RPA-T4 and SK3 respectively, BD Biosciences), PE-CY7- or BV421-labeled CD25 (clone 2A3, BD Biosciences), BV650-labeled CD127 (clone A019D5, Biolegend), APC-H7-labeled CD45RA (clone HI100, BD Biosciences), PerCP-Cy5.5-labeled CD8 (clone SK1, BD Biosciences), PE-CF594- or Alexa Fluor 647-labeled Foxp3 (clone 259D/C7, BD Biosciences), BV421-labeled CTLA-4 (clone BNI3, BD Biosciences), FITC-labeled Ki67 (clone 20Raj1; eBiosciences), APC-labeled Helios (clone 22F6, Biolegend), PE-labeled Tbet (clone ebio4B10; eBiosciences), FITC-labeled CD14 (clone M5E2; BD Biosciences), LIVE-DEAD® Fixable yellow or near-IR dead cell stain kit (ThermoFisher Scientific), and the BD Transcription Factor Buffer set. Acquisition of cells was performed on a BD LSR Fortessa and flow cytometric sorting was done using a BD FACS Aria II. Data was analysed using DIVA software (version 8.02; BD Biosciences).
Live dead fixable yellow or near ir dead cell stain kit
Manufactured by Thermo Fisher Scientific
The LIVE-DEAD® Fixable yellow or near-IR dead cell stain kit is a fluorescent dye-based kit used to detect and distinguish live and dead cells in a sample. The kit provides a simple and effective method for identifying and quantifying the proportion of dead cells in a population.
Automatically generated - may contain errors
2 protocols using live dead fixable yellow or near ir dead cell stain kit
1
Comprehensive Analysis of Treg Subsets
2
Cryopreserved OPSCC Myeloid Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryopreserved single cell OPSCC tumor samples were thawed and used for myeloid cell subset determination by flow cytometry. Cryopreserved healthy donor PBMCs were thawed, depleted of CD3+ T and CD19+ cells by the use of CD3-microbeads (Miltenyi Biotec), subsequently CD19 pan B dynabeads (Thermo Fisher Scientific) and then used for myeloid cell sorting by flow cytometry. After thawing and/or CD3/CD19 depletion, samples were stained with the LIVE-DEAD Fixable yellow or near-IR dead cell stain kit (Thermo Fisher Scientific) for 20 min at room temperature. Following washing, Fc receptors were blocked by incubating for 10 min on ice with phosphate buffered salt (PBS)/0.5% bovine serum albumin (BSA)/10% fetal calf serum (FCS), after which the cells were washed, and stained for 30 min on ice with fluorochrome-conjugated antibodies. Details on antibodies used for myeloid cell subsets determination and flow activated cell sorter (FACS)-sorting of PBMCs are listed in online supplementary table 3 . Acquisition of cells was performed on a BD LSR Fortessa and flow cytometry-based cell sorting was done using a BD FACS Aria III. Data were analyzed using DIVA software (V.8.02; BD Biosciences).
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!