Annexin V and PI (purchased from Sigma-Aldrich)-binding assays were performed to assess apoptosis as described previously [40 (link)]. Annexin V-negative and PI-negative cells were counted as live cells. Apoptosis (A) in a sample was quantified as the proportion of cells that were Annexin V-positive cells, and specific apoptosis was calculated by the following formula: %specific apoptosis = (Atest − Acontrol) × 100/(100 − Acontrol). Thus, by subtracting spontaneous apoptosis (= apoptosis observed in untreated control samples), “specific apoptosis” in the present study indicates specifically induced apoptosis by RHT. For gating stem/progenitor population (CD45+CD34+CD38− cells) in primary AML samples, phycoerythrin (PE)-conjugated anti-human CD45, PE-Cyanine7 (PE-Cy7)-conjugated anti-human CD34, and fluorescein isothiocyanate-conjugated CD38 antibodies and Allophycocyanin-conjugated Annexin V (BD Biosciences, San Jose, CA) were used.
Pe conjugated anti human cd45
Manufactured by BD
Sourced in United States
PE-conjugated anti-human CD45 is a laboratory reagent used to detect and identify CD45-expressing cells in research samples. CD45 is a cell surface glycoprotein found on all human leukocytes. The PE (phycoerythrin) fluorescent dye is conjugated to the anti-CD45 antibody, enabling its use in flow cytometry and other applications that require fluorescent labeling of cells.
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Lab products found in correlation
Pe conjugated anti human cd235a, by BD (2 mentions)
Pe conjugated anti human cd31, by BD (2 mentions)
Allophycocyanin conjugated annexin 5, by BD (1 mentions)
Annexin 5, by Merck Group (1 mentions)
Facsaria, by BD (1 mentions)
Allophycocyanin conjugated anti mouse cd45, by BD (1 mentions)
Pe conjugated anti human cd19, by BioLegend (1 mentions)
Rbc lysis solution, by Qiagen (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
5 protocols using pe conjugated anti human cd45
1
Quantifying Apoptosis in Primary AML Samples
2
Isolation of Epithelial Cells from Normal Tissue
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To assess epithelial cell marker expression on normal tissue, single cells were blocked in phosphate‐buffered saline (PBS) containing 2% FCS, 10% DNase, rat immunoglobulin (Jackson Immunolabs), and antibodies to CD16 and CD32 Fcγ II and III receptors (WEHI Monoclonal Antibody Facility) for 10 min at 4°C. Cells were then incubated with the following antibodies for 25 min at 4°C: PE‐conjugated anti‐human CD31 (BD Pharmingen; clone WM59; 1/40), PE‐conjugated anti‐human CD45 (BD Pharmingen; clone H130; 1/120), PE‐conjugated anti‐human CD235a (BD Pharmingen; clone GA‐R2; 1/120), FITC‐conjugated anti‐human CD236 (EpCAM; Stem Cell Technologies; clone VU‐1D9; 1/40), and APC‐Cy7‐conjugated anti‐human CD49f (integrin a6; clone GoH3; 1/120). Cells were then washed with PBS/2% FCS and resuspended in 7‐AAD (0.2 mg/ml) for live‐cell discrimination. Cells were sorted on a FACSAria flow cytometer (Becton Dickinson). For normal tissue, lineage‐negative (depleted for CD45, CD31, CD235a lineage‐positive cells), epithelial cells (EpCAM+CD49f– + EpCAM+CD49f+ + EpCAMCD49f+) were sorted.
3
Evaluating Leukemic Cell Engraftment in NOG Mice
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Human leukemic cell engraftment was evaluated by flow cytometry analysis of BM aspirates from transplanted NOG mice after 2 or 4 weeks. The leukemic cell surface markers, human CD19 and CD3, were used to identify B‐ALL and T‐ALL samples, respectively. Antibodies used in this study were as follows: allophycocyanin‐conjugated anti‐mouse CD45 (BD Pharmingen), PE‐conjugated anti‐human CD45 (BD Pharmingen), PE‐conjugated anti‐human CD19 (Biolegend), and fluorescein isothiocyanate‐conjugated anti‐human CD3 (BD Pharmingen).
4
Peripheral Blood RBC Lysis and Cytometry
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About 0.5 ml peripheral blood was harvested and subjected to RBC lysis using RBC lysis solution (Qiagen, Valencia, CA, USA). Cells were stained with the PE-conjugated anti-human CD45 (BD, Franklin Lakes, NJ, USA) and FITC conjugated Mouse CD45.1 antibodies and then analyzed on BD FACSCanto II instruments.
5
Multicolor Flow Cytometry of Cell Subsets
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Cells were blocked with rat immunoglobulin (Jackson ImmunoLabs) and antibody to Fc receptor–binding inhibitor (eBioscience) before incubation with the following primary antibodies: phycoerythrin (PE)–conjugated anti-human CD31 (BD Pharmingen), PE-conjugated anti-human CD45 (BD Pharmingen), PE-conjugated anti-human CD235a (BD Pharmingen), BV650-conjugated anti-human epithelial cell adhesion molecule (EpCAM) CD326 (BioLegend), and biotin-conjugated anti-human ITGA6 (eBioscience). Where required, cells were incubated with allophycocyanin-Cy7–conjugated streptavidin (BD Pharmingen). Cells were either stained with 4′,6-diamidino-2-phenylindole (DAPI) for viability or fixed with 1% paraformaldehyde and stained with the Zombie Aqua Fixable Viability Kit (BioLegend). Viable cells were sorted on a FACSAria flow cytometer (Becton Dickinson). Data were analyzed using FlowJo software (Tree Star).
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