Purified T cells were stimulated as previously described and GM-CSF was measured using the GM-CSF Secretion Assay Enrichment and Detection Kit (PE, Miltenyi, 130-105-760). The manufacturer’s instructions were modified to a 96-well format with final volumes of 200 μl per well. Following the GM-CSF kit protocol, cells were additionally stained with CD4 (clone SK3, BD, BV480, 566104), CD8 (clone SK1, BD, BUV805, 564912), CD3 (clone UCTH1, BD, BB630, 624294), HLA-DR (clone G46-6, BD, BV786, 564041), CD16 (clone 3G8, BD, BV750, 624380), CD45 (clone 2D1, BD, BUV563, 624284), CD69 (clone FN50, BD, APC-H7, 560737), CCR7 (clone G043H7, Biolegend, BV650, 353134), CD45RA (cloneHI100, BD, PE, 561883), CD25 (clone 2A3, BD, PE-Cy7, 335789), CD14 (M5E2, BD, BV570, 624298), CD56 (clone NCAM16.2, BD, PE-Cy5, 624350) and live-dead dye FVS575V (BD, BV570, 565694). Subsequently, cells were fixed and permeabilized in Foxp3/transcription factor staining buffer as previously described, and stained with the above-mentioned metabolic antibodies, before acquiring on the X-30 FACSymphony.
Clone 2d1
Manufactured by BD
Sourced in United States
The Clone 2D1 is a laboratory instrument designed for cell culture applications. It provides a controlled and monitored environment for the growth and maintenance of cell lines. The core function of the Clone 2D1 is to maintain optimal temperature, humidity, and gas composition for cell culture.
Automatically generated - may contain errors
Lab products found in correlation
Clone g043h7, by BioLegend (1 mentions)
Cd3 clone ucth1, by BD (1 mentions)
Clone ncam16, by BD (1 mentions)
Clone hb 7, by BD (1 mentions)
Facslyric, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Cd45 percp cy5, by BD (1 mentions)
4 protocols using clone 2d1
1
Monitoring T cell Activation and Phenotype
2
Flow Cytometric Immune Cell Analysis
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Flowcytometric measurements were carried out on a BD FACSLyric (Becton Dickinson, BD Life Sciences, San Jose, CA, 95131, USA) by measuring 150,000 events of stained sample. Staining was performed by incubating 100 μL EDTA-anticoagulated peripheral blood with anti-CD19 PE (Becton Dickinson, Clone SjJ25C1, BD Life Sciences, San Jose, CA, 95131, USA), anti-CD20 APC (Becton Dickinson, Clone L27, BD Life Sciences, San Jose, CA, 95131, USA), anti-CD45 PerCP (Becton Dickinson, Clone 2D1, BD Life Sciences, San Jose, CA, 95131, USA), and anti-CD38 FITC (Becton Dickinson, Clone HB-7, BD Life Sciences, San Jose, CA, 95131, USA) for 10 min at room temperature in the dark. Cells were then lysed for 10 min with 1× BDlysing solution, washed once with BDFACS Flow Solution, and resuspended therein for immediate acquisition. FCS files were analyzed with FACSSuite. B-cells were identified as CD19 expressing lymphocytes, excluding Plasmablasts/Plasmacells, based on their CD38 expression. CD19 positive B-cells were evaluated for CD20 co-expression.
3
Neutrophil Characterization by Flow Cytometry
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Phycoerythrin (PE)-conjugated antibody specific for CD177 (clone MEM-166; BD Biosciences, Franklin Lakes, NJ, USA) and fluorescein isothiocyanate (FITC)-conjugated antibody specific for human CD45 (clone 2D1; BD Biosciences) were used for neutrophil staining. Isotype control antibodies including PE-mouse IgG1, κ (clone MOPC-21; BD Biosciences) and FITC-mouse IgG1, κ (clone MOPC-31C; BD Biosciences) were used to determine non-specific binding of antibodies. All antibodies were titrated before use to determine the concentration giving minimum saturation binding.
4
Flow Cytometric Characterization of Hematopoietic and Mesenchymal Stem/Progenitor Cells
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For flow cytometric analysis of HSPCs, suspension cells were harvested, treated with FcR blocking reagent (Miltenyi Biotec) and stained with CD45‐peridinin chlorophyll‐cyanin 5.5 (CD45‐PerCP‐CY5.5; mIgG1, Clone TU116; BD Biosciences), or CD34‐allophycocyanin (CD34‐APC; mIgG2a, Clone AC136) and CD133‐phycoerythrin (CD133‐PE; mIgG2b, Clone AC133) antibodies or matched isotype control antibodies (Miltenyi Biotec). For analysis of hBMSC, cells were trypsinized and then stained with either CD31‐PE (mIgG1, Clone WM9), CD44‐PE (mIgG1, Clone 515), CD14‐PE‐CY7 (mIgG2a, Clone M5E2), CD29‐PE (mIgG1, Clone MAR4), CD45‐PerCP (mIgG1, Clone 2D1; all BD Biosciences), CD90‐fluorescein isothiocyanate (CD90‐FITC; mIgG1, 5E10), CD105‐FITC (mIgG1, 166707), CD166‐PE (mIgG1, Clone 105902; all R&D Systems, Abingdon, UK) or CD34‐APC (mIgG2a, AC136; Miltenyi Biotech) antibodies or matched isotype controls from the same manufacturer. For analysis of bone marrow endothelium, mouse bones were harvested 4 d post‐irradiation, crushed and then stained with a panel of haematopoietic/endothelial markers exactly as described previously by Hooper et al (2009 ). In these experiments DAPI was used to distinguish dead and viable cells. Data were acquired using a BD LSR II flow cytometer and analysed using the FACS Diva software (BD Biosciences).
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