Cells were stained with cell-surface antibodies against CD3-allophycocyanin-Cy7 (clone SK7, BD Pharmingen, San Jose, USA), CD4-phycoerythrin (clone RPA-T4, BD Pharmingen), CD8-fluorescein isothiocyanate (clone RPA-T8; BD Pharmingen) and CD33-allophycocyanin (clone WM53, BD Pharmingen). Cells were washed twice with flow cytometry staining buffer and re-suspended in Pre-Sort buffer (BD Biosciences). 7-AAD viability dye (eBioscience, San Diego, USA) was used to gate live cells. BD FACSAria III SORP cell sorter on BD FACSDiva software (BD Biosciences) was used for sorting pure CD4+ (7AAD–CD3+CD4+CD8–CD33–), CD8+ (7AAD–CD3+CD4–CD8+CD33–) and CD33+(7AAD–CD3–CD4–CD8–CD33+) populations. Applicable measures were taken to ensure minimal sorter-induced cell stress (SICS). Data analyses were performed on FlowJo V10 software (FlowJo, Ashland, USA).
Cd8 fluorescein isothiocyanate clone rpa t8
Manufactured by BD
Sourced in United States
CD8-fluorescein isothiocyanate (clone RPA-T8) is a fluorescently-labeled monoclonal antibody used for the detection and quantification of CD8+ T cells in flow cytometry applications. It binds specifically to the CD8 cell surface antigen expressed on a subset of T cells.
Automatically generated - may contain errors
Lab products found in correlation
7 aad viability dye, by Thermo Fisher Scientific (2 mentions)
Pre sort buffer, by BD (2 mentions)
Cd3 allophycocyanin cy7 clone sk7, by BD (2 mentions)
Cd4 phycoerythrin clone rpa t4, by BD (2 mentions)
Facsaria 3 sorp cell sorter on facsdiva software, by BD (1 mentions)
Cd33 allophycocyanin clone wm53, by BD (1 mentions)
Facsaria 3 sorp cell sorter, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
2 protocols using cd8 fluorescein isothiocyanate clone rpa t8
1
Fluorescence-Activated Cell Sorting of Immune Cell Subsets
2
Isolation of CD4+ and CD8+ TILs
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Single cell suspensions from TT specimens were resuspended in 100 µL of flow cytometry staining buffer (PBS with 1% FCS and 0.1% sodium azide). An FcR Blocking Reagent (Miltenyi Biotec) was used to block Fc receptors (FcR) and a 7-AAD viability dye (eBioscience, San Diego, CA, USA) was used to gate live cells. Cells were stained with surface antibodies against CD3-allophycocyanin-Cy7 (clone SK7, BD Pharmingen, San Jose, CA, USA), CD4-phycoerythrin (clone RPA-T4, BD Pharmingen), and CD8-fluorescein isothiocyanate (clone RPA-T8; BD Pharmingen). Cells were washed twice with flow cytometry staining buffer prior to re-suspension in Pre-Sort buffer (BD Biosciences, Oxford, UK). A BD FACSAria III SORP cell sorter on BD FACSDiva software (BD Biosciences) was used for sorting pure CD4+ (7AAD−CD3+CD4+CD8−), and CD8+ (7AAD−CD3+CD4−CD8+) TILs. Relevant procedures were followed to ensure minimum sorter-induced cell stress (SICS), as previously described [18 (link)]. Flow cytometric analyses were performed on FlowJo V10 software (FlowJo, Ashland, OH, USA).
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