For the flow cytometry analysis, EDTA anticoagulated samples were used. The bone marrow samples were strained using special tubes (5 ml polystyrene round-bottom tube with cell-strainer cap, cat.no:352235, BD Biosciences). For the staining of the cells (BM-MNC or PBMC), the following markers were used, according to the manufacturers protocol: CD19 PC7 (cat. no.: BCI-F-IM3628, Beckman Coulter Company), CD20 APC-H7, (cat.no.: 561172, BD Biosciences), CD27 PerCP-Cy5.5 (cat.no.: 560612, BD Biosciences), CD28, PerCP-Cy5.5 (cat.no.: 337181, BD Biosciences), CD38 APC (cat.no.: 345807, BD Biosciences), CD45 Krome Orange (cat.no.: BCI-F-IM36294, Beckman Coulter Company), CD56 FITC (cat. no:345811, BD Biosciences), CD81 APC-H7 (cat. no:656647, BD Biosciences), CD117 PE, (cat. no.: 332785, BD Biosciences), CD138 VioBlue (cat.no.:130-119-843, Miltenyi Biotec), Kappa FITC (cat. no.:349516, BD Biosciences) (intracellular staining), Lambda PE (cat. no.:349516, BD Biosciences) (intracellular staining). For the intracellular staining, BD Intrasure Kit was used (cat.no.: 641778 BD Biosciences). The acquisition of the samples was done on a FACS Canto II cytometer (BD Biosciences), and the analyses were done with the Infinicyt (ver.2.0.2c, Cytognos S.L.) software.
Cd20 apc h7
Manufactured by BD
Sourced in United States
CD20 APC-H7 is a fluorescently-labeled antibody that binds to the CD20 antigen on the surface of B cells. It is used for the identification and analysis of B cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 pe, by BD (3 mentions)
Cd38 alexa fluor 700, by BD (3 mentions)
Cd19 apc, by BD (3 mentions)
Intrasure kit, by BD (2 mentions)
Igd pe, by BD (2 mentions)
Cd10 pe cy7, by BD (2 mentions)
Cd11a bv510, by BD (2 mentions)
Facscanto 10, by BD (2 mentions)
Syto41, by Thermo Fisher Scientific (2 mentions)
Cd45 percp, by BD (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Spa 896, by Enzo Life Sciences (2 mentions)
Cd81 apch7, by BD (1 mentions)
Cd117 pe, by BD (1 mentions)
Lambda pe, by BD (1 mentions)
Cd38 apc, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Cd45 krome orange, by Beckman Coulter (1 mentions)
Cd27 percp cy5, by BD (1 mentions)
Cd28 percp cy5, by BD (1 mentions)
7 protocols using cd20 apc h7
1
Flow Cytometry Analysis of Bone Marrow and Peripheral Blood Cells
2
Phenotypic Analysis of Cryopreserved PBMCs
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Cryopreserved PBMCs were thawed and washed twice with 10 mL of FACS buffer (1 x PBS containing 2% FBS and 1 mM EDTA) and resuspended in 100 uL of 1x PBS containing Zombie UV live/dead dye at 1:200 dilution (BioLegend, 423108) and incubate at room temperature for 15 minutes. Following washing, cells were incubated with an antibody cocktail for 1 hour protected from light on ice. The following antibodies were used: IgD PE (BD Biosciences, 555779), IgM PerCP-Cy5.5 (BioLegend, 314512), CD20 APC-H7 (BD Biosciences, 560734), CD27 PE-Cy7 (BioLegend, 302838), CD14 PE/Dazzle™ 594 (BioLegend, 301852), CD16 BV605 (BioLegend, 302040), IgG BV650 (BD Biosciences, 740596), CD3 BUV737 (BD Biosciences, 612750) and Alexa Fluor 488-labeled Wuhan spike (SinoBiological, 40589-V27B-B), and BV421 labeled Omicron Spike (SinoBiological™, 40589-V49H3-B). All antibodies were used as the manufacturer’s instruction and the final concentration of each probe was 0.1 ug/ml. Cells were washed twice in FACS buffer and immediately acquired on a BD FACS Aria III for acquisition and FlowJo for analysis.
3
Multicolor Flow Cytometry for MRD Detection
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Multicolor flow cytometry analysis of BM was performed at the day of diagnosis to assess leukemic-associated immunophenotype (LAIP) of blasts cells. A following antibodies were used for cell immunophenotyping: CD34-PE, CD45-PerCP, CD10-PE-Cy7, CD19-APC, CD38-AlexaFluor-700, CD20-APC H7, CD11a-BV510 (BD Biosciences, Waltham, MA, USA), CD58 (Beckman Coulter, Brea, CA, USA). On the day 15th and 33rd BM was analyzed using the same panel of antibodies to determine MRD. To the appropriate amount of BM (106 cells) antibodies listed above were added, samples were incubated for 20 min at room temperature in darkness. Erythrocytes were lysed for 10 min at room temperature in darkness with lysing solution (BD FACS Lysing Solution, Becton Dickinson Biosciences, San Jose, CA, USA), washed twice in PBS, and finally resuspended in 200 μL of PBS. For distinguishing nucleated cells, samples were stained with Syto®41 (Thermo Fisher Scientific, Waltham, MA, USA). FACS analysis was done using FACSCanto or FACSCanto10 with FACSDiva Sorfware v. 8.1 (Becton Dickinson Biosciences, San Jose, CA, USA).
4
Intracellular HO-1 Expression in BM
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HO-1 expression was assessed in BM of 11 patients upon diagnosis. Then in patients in whom, after routine MRD staining, there was a sufficient material for additional staining, an intracellular HO-1 staining was performed (6 children at day 15, 3 children at day 33). In this purpose 106 of bone marrow cells were stained with CD34-PE, CD45-PerCP, CD10-PE-Cy7, CD19-APC, CD38-AlexaFluor-700, CD20-APC H7, CD11a-BV510 (BD Biosciences, San Jose, CA, USA), lysed, fixed, and then permeabilized using a BD Intrasure Kit, according to the manufacturer’s instructions. After permeabilization step, cells were incubated with primary antibodies recognizing HO-1 (rabbit polyclonal, SPA 896, Enzo, Warszawa, Poland), washed twice, and stained with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Life technologies, HITACHI, Tokyo, Japan). After two washing steps, samples were stained with Syto®41 (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using FACSCanto10 with FACSDiva Sorfware v 8.0.1 (Becton Dickinson Biosciences, San Jose, CA, USA).
5
Flow Cytometric Immunophenotyping of PBLs
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Following isolation, PBLs were washed once in 1X PBS +1% FBS, resuspended in 150 μL 1X PBS +1% FBS containing the antibody cocktail (anti- CD3-AF488 (BD Biosciences, clone SP34-2), CD4-AF700 (BD Biosciences, clone SK3), CD8-BV421 (BD Biosciences, clone SK1), CD20-APC-H7 (BD Biosciences, clone 2H7), CD14-APC (BD Biosciences, clone m5e2), and CD16-PeCy7 (BD Biosciences, clone 3g8), and incubated for 20 min at 4°C in the dark. Cells were then washed twice with 1 mL 1X PBS +1% FBS, centrifuged for 5 min at 530 g, and fixed with 150 μL 4% methanol-free formaldehyde for 15 min at 4°C in the dark. Finally, 1 mL 1X PBS was added and the fixed cells centrifuged at 1,470 g for 5 min. The final pellets were resuspended in 500 μL 1X PBS and the data acquired using a Gallios flow cytometer (Beckman Coulter) and analyzed using Flowjo V.10.8 (Treestar).
6
Immunofluorescence Staining of Hematopoietic Stem Cells
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In order to prepare immunofluorescence slides of certain hematopoietic stem or progenitor cell subsets, characterized elsewhere [68 (link),107 (link)], normal BM samples were stained using following primary antibodies: Lin-FITC, CD90-PE, CD34-APC, CD38-AlexaFluor700, CD45-APC-H7, CD45RA-PE-Cy-7 or CD38-FITC, CD34-PE, CD19-APC, CD20-APC-H7 (BD Biosciences, San Jose, CA, USA). Cells of defined immunophenotype were sorted using MoFlo-XDP cell sorter (Beckman Coulter, Brea, CA, USA) into 20 mL PBS drops on poly-L-lysine coated slides. After settling, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X100. Afterwards, samples were incubated with 0.25% glycine for 30 min, followed by blocking with 3% BSA in PBS for 1 h. Subsequently, samples were stained overnight at 4 °C with primary antibodies recognizing HO-1 (rabbit polyclonal, SPA 896, Enzo, Warszawa, Poland) in a moisture chamber. After 5 washing steps, slides were incubated with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Life technologies, Carlsbad, CA, USA) and DAPI for 1 h in darkness. After the next 5 washing steps, cells were analyzed using a Zeiss confocal microscope with ZEN Software (Zeiss, Oberkochen, Germany).
7
Multiparametric Flow Cytometry Analysis of PBMCs
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PBMCs were resuspended in PBS, containing 0.5% w/v BSA and 0.01% sodium azide. PBMCs were incubated with saturating concentrations of fluorescently labeled conjugated mAbs. Analysis of cells was performed using a FACSCanto‐II flowcytometer and FlowJo software (version 9.1 and 10) and for the methods of flow cytometry we adhered to the ‘Guidelines for the use of flow cytometry and cell sorting in immunological studies’ 38 . The following mAbs were used for flow cytometry: CD3 Alexa Fluor 700 [557943], CD4 PE‐Cy7 [348809], CD8 PerCP‐Cy5.5 [341050], CD19 Alexa Fluor 700 [557921], CD19 PerCP‐Cy 5.5 [332780], CD20 APC‐H7 [641414], CD20 PerCP‐Cy5.5 [332781], CD25 APC [340907], CD27 APC [337169], CD38 PE [345806], CD38 PE‐Cy7 [335825], HLA‐DR FITC [347400], and IgD PE [555779] from BD (San Jose, USA); CD3 Alexa 700 [56‐0038‐41], CD19 Alexa Fluor 700 [56‐0199‐42], and CD27 APC‐eFluor 780 [47‐0279‐42] from eBioscience (San Diego, USA); and CD27 FITC [M1764] from Sanquin (Amsterdam, the Netherlands). To assess lymphocyte viability TO‐PRO‐3 iodide [T3605] was used (Thermo Fisher Scientific, Massachusetts, USA).
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