Commercial antibodies and staining reagents originated from BioLegend: CD45.1-FITC, CD45.2-APC, CD38-PE-Cy7, CD4-PerCP, CD8a-PerCP-Cy5.5, IFNAR1 biotin, and streptavidin conjugated to BV786; from BD Biosciences: CD16/CD32, CD138-BV650, B220-PacBlue, CD95 (APO-1/Fas)-PE; from ThermoFisher Scientific: GL7-A488, Ki67-efluor660 and viability dye fixable live/dead stain eFlour780. The 9D11 hybridoma expressing anti-idiotypic monoclonal IgG1 antibody, clone 9D11 (41 (link)), was kindly provided by Elisabeth Alicot, Boston Children’s Hospital, and was conjugated with iFluor647 succinimidyl ester (AAT Bioquest) in-house.
Cd8a percp cy5
Manufactured by BD
Sourced in United States
The CD8a-PerCP-Cy5.5 is a fluorescent-conjugated antibody that binds to the CD8a surface marker. It is used for the identification and analysis of CD8-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 percp, by BD (1 mentions)
Cd16 cd32, by Thermo Fisher Scientific (1 mentions)
Cd45.2 apc, by BD (1 mentions)
Cd45.1 fitc, by BioLegend (1 mentions)
Cd38 pe cy7, by BD (1 mentions)
Cd3 percp cy5, by BD (1 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Biowest (1 mentions)
Tnf α apc, by BD (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd4 percp cy5, by BD (1 mentions)
Ifnγ brilliant violet 605, by BD (1 mentions)
Il 4 brilliant violet 421, by BD (1 mentions)
Il 13 fitc, by BD (1 mentions)
Il 17a apc cy7, by BD (1 mentions)
Lyric flow cytometer, by BD (1 mentions)
Siglecf percp cy5, by BD (1 mentions)
Il 17f pe, by BD (1 mentions)
Gr 1 percp cy5, by BD (1 mentions)
F4 80 pe, by BD (1 mentions)
5 protocols using cd8a percp cy5
1
Multiparameter Immune Cell Profiling
2
Quantifying Cytokine Production in Splenic ILCs
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The number of ILCs producing various cytokines in the spleen was measured using flow cytometry (FCM). Analysis was performed on 5–8 animals per group. The spleen was crushed in a petri dish, and the cells were filtered through a mesh and incubated with ACK Lysing Buffer (Thermo Fisher Scientific, Waltham, MA, USA) to lyse the red blood cells. Mononuclear cells were isolated and purified by density gradient centrifugation. The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used to exclude apoptotic and necrotic cells. The cultured mononuclear cells were stained with surface antibodies: CD45R-PerCp-Cy5.5, CD3-PerCp-Cy5.5, CD4-PerCp-Cy5.5, FceRI-PerCp-Cy5.5, CD8a-PerCp-Cy5.5, Gr-1-PerCp-Cy5.5, Siglec-F-PerCp-Cy5.5 (BD Biosciences, Franklin Lakes, NJ, USA) in cell surface staining buffer containing 0.1 M phosphate-buffered saline and 2% FCS (Biowest, Nuaillé, France) [61 (link),62 (link)], and then stained with IFNγ-Brilliant Violet 605, TNFα-APC, IL-4-Brilliant Violet 421, IL-13-FITC, IL-17A-APC-Cy7, and IL-17F-PE antibodies (BD Biosciences). The expression patterns of inflammatory cytokines were analyzed using a BD Lyric flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (v10.9.0) (Tree Star Inc., Ashland, OR, USA).
3
Lung Cell Immunophenotyping by Flow Cytometry
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The lung cell suspensions were centrifuged at 300×g for 5 minutes to obtain the cell pellet. Approximately 1 × 106 cells per sample were stained with Fixable Near-IR Live/Dead (Invitrogen, L34975) and monoclonal antibodies (CD45-BV510 [BD, 2154345], CD4-PE-CY7 [BD, 552775], CD8a-PerCP-Cy5.5 [BD, 551162], F4/80-PE [BD, 2165051]) for 30 minutes at 4°C. The stained cells were fixed in 1.5% paraformaldehyde for 5 minutes and analyzed using a Cytoflex S flow cytometer (Beckman). The data were analyzed with FlowJo software.
4
Flow Cytometry Analysis of Immune Cell Subsets
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SVF pellets were washed with PBS/2% FBS, incubated with Fc Block (BD Bioscience, San Jose, CA, USA) for 15 min and then stained with the following conjugated antibodies (30 min at 4 °C in the dark): CD45-APC-Cy7, CD3-APC, CD19-APC, NK1.1-APC, F4/80-PE-Cy7, CD11b-PerCP-Cy5.5 (all from BD Bioscience), and TER119-APC and CD11c-PE (eBioscience, San Diego, CA, USA) for ATM, CD45-APC-Cy7, CD19-PE, CD4-V500, CD8a-PerCP-Cy5.5, Gr1-APC (BD Bioscience), and CD3-eFlour450, NK1.1-PE-Cy7, TER119-APC, F4/80-APC (eBioscience) for lymphocyte population. After incubation, cells were washed and resuspended in PBS/2% FBS and then analyzed by FACS Canto II (BD Biosciences) and FlowJo software (version 10, Tree Star Inc., Ashland, OR, USA). As described previously [25 (link)], ATM were defined as CD45+, CD3−, CD19−, NK1.1−, TER119−, CD11b+ and F4/80+. T cells (CD19−CD3+NK1.1−), B cells (CD19+CD3−), and NK cells (CD19−CD3−NK1.1+) were gated after excluding the myeloid lineage (Gr1+, F4/80+) and red blood cells (TER119+) among immune cells (CD45+).
5
Multiparametric Flow Cytometry Analysis
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Single‐cell suspensions were obtained from cultured cells or tumor tissues. The tumors were enzymatically digested in 1 mg mL−1 collagenase I and 1 mg mL−1 collagenase IV. Fixable Viability Stain 700 (564997, BD) was used to exclude dead cells. The following antibodies were used for surface staining: CD3e‐APC‐Cy™7 (557596, BD), CD8a‐PerCP‐Cy™5.5 (551162, BD), CD335‐BV421 (562850, BD), CD11b‐FITC (557396, BD), CD80‐APC (ab95549, Abcam), CD86‐PerCP‐Cy5.5 (105027, Biolegend), Gr‐1‐APC (108411, Biolegend) and CD11c‐FITC (117306, Biolegend). After being stained at room temperature in the dark, the cells were fixed with 4% methanol, permeabilized with 0.5% Triton, and intracellularly stained with Foxp3‐Alexa Fluor 647 (560401, BD). A BD FACSymphony A5 instrument was utilized for flow cytometry, and NovoExpress Software (Tree Star) was employed to analyze the data. Cell populations were identified as follows: CD4+ T cells: live/CD45+/CD3+/CD4+/FoxP3−; CD8+ T cells: live/CD45+/CD3+/CD8+; Treg cells: live/CD45+/CD3+/CD4+/FoxP3+; natural killer (NK) cells: live/CD45+/CD3−/NKP46+; MDSC cells: live/ CD11b+/Gr1+ and DCs: live/CD45+/CD3−/CD11c+.
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