Spleen and bone marrow cells (4 x 106) were disaggregated into single-cell suspensions, treated with RBC lysis buffer (eBioscience) then stimulated with PMA (25 ng/ml) and ionomycin (1 µg/ml) (Sigma). After stimulation, the cells were stained with NK-1.1-allophycocyanin (PK136), CD3-AlexaFluor700 (17A2), CD8α-FITC (53–6.7) and CD4-PE-CF594 (RM4–5) (all from BD Biosciences) for 30 min. The cells were permeabilized in Perm/Fix buffer (BD Biosciences) then stained with IFNγ-PE (XMG1.2) (BD Biosciences). All cells were fixed in 1% paraformaldehyde before analysis with an LSR-II flow cytometer (BD Biosciences). Up to 500,000 events per sample was captured for data analysis using the FlowJo software (Treestar). Isotype controls (BD Biosciences) were used for gating.
Cd4 pecf594 rm4 5
Manufactured by BD
Sourced in United Kingdom, United States
The CD4 PECF594 (RM4-5) is a laboratory instrument used for the detection and quantification of CD4+ T cells. It is a flow cytometry-based tool that utilizes fluorescent-labeled antibodies to identify and analyze the expression of the CD4 surface marker on cells. The core function of this product is to provide researchers and clinicians with a reliable method for assessing the levels of CD4+ T cells in biological samples.
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Lab products found in correlation
Ifnγ pe xmg1.2, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Isotype control, by BD (1 mentions)
Fix perm buffer, by BD (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Live dead fixable near ir stain, by Thermo Fisher Scientific (1 mentions)
Cd3e apc 145 2c11, by BioLegend (1 mentions)
Red 780, by Cytek Biosciences (1 mentions)
Mouse th1 th2 th17 phenotyping kit, by BD (1 mentions)
Ki67 b56, by BD (1 mentions)
Fatp2, by Abcam (1 mentions)
Goat anti rabbit cy3, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit af488, by Abcam (1 mentions)
4 protocols using cd4 pecf594 rm4 5
1
Multiparametric Flow Cytometry of Immune Cells
2
Detailed Flow Cytometric Immunophenotyping
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Flow cytometric analysis was performed using the following antibodies: CD4 PECF594 (RM4‐5) from BD Biosciences (Oxford, UK) and CD8 peridinin chlorophyll (PerCP) (SK1), CD45RA BV605 (HI100), CD27 BV421 (O323), CCR7 phycoerythrin cyanin 7 (PECy7) (G043H7), CXCR2 fluorescein isothiocyanate (FITC) (5E8) and CX3CR1 (K0124E1) from BioLegend (London, UK), together with a live/dead fixable near‐IR stain (Invitrogen, Basingstoke, UK). A total of 1 × 106 PBMCs were incubated with antibody for 15 min at room temperature, after which time they were fixed in 1% paraformaldehyde in phosphate‐buffered saline (PBS). All samples were run using an LSR II (BD Biosciences) and analysed using FlowJo software (Treestar, Ashland, OR, USA). The gate strategy can be seen in Supporting information, Fig. S1 .
3
Multiparameter T Cell Phenotyping
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Splenocytes were resuspended in a buffer of 1% BSA, 2% FBS, 0.03% sodium azide, and 2 mM EDTA in PBS for staining with CD8b-Alexa Fluor 488 (H35-17.2, Invitrogen), CD4-PE-CF594 (RM4-5, BD), CD62L-PE-Cy7 (MEL-14, Tonbo Biosciences, San Diego, USA), CD44-Brilliant Violet 421 (IM7, Biolegend), CD3e-APC (145-2C11, Biolegend), and/or ghost dye (Red 780, Tonbo Biosciences) and were fixed in 2% paraformaldehyde in PBS after staining. Helper T cell characterization was performed using a mouse Th1/Th2/Th17 Phenotyping Kit (BD Biosciences, San Jose, USA) per the manufacturer's instructions. Samples were acquired using a LSRII machine, and data were analyzed using FlowJo software (BD Biosciences).
4
Flow Cytometric Analysis of T Cell Subsets
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Flow cytometric analysis was performed using the following antibodies: CD4 PECF594 (RM4‐5) from BD Biosciences and CD8 PerCP (SK1), CD45RA BV605 (HI100), CD27 BV421 (O323), CD28 BV785 (CD28.2), CCR7 PECy7 (G043H7) and CD36 APCCy7 (5‐271) from BioLegend. FATP2 (Abcam) and FATP3 (Atlas antibodies) were measured in conjunction with goat anti‐rabbit AF488 (Abcam). Cortactin expression was assessed using rabbit anti‐human cortactin antibody (PA5‐27134; Life Technologies) stained in conjugation with goat anti‐rabbit Cy3 (Life Technologies). PGC1α (3G6) and p‐p53 (16G8) both from Cell Signaling, and Ki67 (B56; BD Bioscience) were assessed by intracellular staining using solution AB (Thermo Fisher) and goat anti‐rabbit AF488 (Abcam). All samples were run using an LSR II (BD Biosciences) and analysed using FlowJo software (Treestar).
Magnetic beads were used to isolation of CD8+ and CD4+ T cells (Miltenyi Biotec) according to the manufacturer's instructions. The purity of T cell subsets was assessed by flow cytometry.
Magnetic beads were used to isolation of CD8+ and CD4+ T cells (Miltenyi Biotec) according to the manufacturer's instructions. The purity of T cell subsets was assessed by flow cytometry.
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