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Flow cytometry staining buffer solution

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The Thermo Fisher Scientific Flow Cytometry Staining Buffer Solution is a buffered formulation designed for cell surface staining and analysis by flow cytometry. It maintains the viability and integrity of cells during the staining process.

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Lab products found in correlation

Goat anti human secondary antibody, by Thermo Fisher Scientific (2 mentions) Lsrfortessa cell analyzer, by BD (2 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Facscaliburtm flow cytometer, by BD (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Lsr fortessa cytometer, by BD (1 mentions) Facscantotm, by BD (1 mentions) Human msc phenotyping kit, by Miltenyi Biotec (1 mentions) Nhs fitc, by Thermo Fisher Scientific (1 mentions)

5 protocols using flow cytometry staining buffer solution

1

Isolation and Characterization of PBMC-derived Macrophages

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Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) density gradient centrifugation method. Briefly, Diluted blood sample (4 ml) was layered on Ficoll-Paque Plus (3 ml) and centrifuged at 400 ×g for 30 minutes at 20°C. Mononuclear cell layer was transferred to a clean centrifuge tube, subsequently, washed by 3 volumes of RPMI 1640 medium (Gibco, USA) and centrifuged at 100 ×g for 10 minutes. After washed one more time with RPMI 1640, cells were resuspended in flow cytometry staining buffer solution (eBioscience, San Diego, CA) and stained with fluorescent antibodies against CD14 (APC), CD11c, and CD163 (BD Biosciences, Heidelberg, Germany) to differentiate M1 from M2 macrophages. The stained cells were assayed by flow cytometry on a FACS CaliburTM flow cytometer (BD Biosciences, Germany) and data were analyzed with FlowJo software.
Ma Y., Ye Y., Zhang J., Ruan C.C, & Gao P.J. (2019). Immune imbalance is associated with the development of preeclampsia. Medicine, 98(14), e15080.
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2

CD30 Antibody Binding Assay

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The binding of the antibody to CD30 was assessed by flow cytometry in H460, H358, and A549 cells. Cells were harvested, washed with cold PBS, and resuspended in Flow Cytometry Staining Buffer Solution (eBioscience, USA) at a concentration of 1 × 106 cells/mL. Cells were incubated for 60 min on ice with either BV or Df-BV (at 5 or 25 μg/mL) and washed. Cells were then incubated with 3 μg/mL of goat anti-human AF488 secondary antibody (Thermo-Fisher Scientific, USA) for 30 min on ice. After washing, cells were analyzed using a LSRFortessa cytometer (BD Biosciences, USA) and mean fluorescence intensities were quantified using FlowJo (Tree Star, USA).
Kang L., Jiang D., Ehlerding E.B., Barnhart T.E., Ni D., Engle J.W., Wang R., Huang P., Xu X, & Cal W. (2018). Noninvasive Trafficking of Brentuximab Vedotin and PET Imaging of CD30 in Lung Cancer Murine Models. Molecular pharmaceutics, 15(4), 1627-1634.
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3

Characterization of hPDC Stemness Markers

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hPDCs cultured in monolayer or aggregates were characterized for expression of stemness markers (CD73, CD90, and CD105) by flow cytometry using human MSC Phenotyping kit (Lot# 130-095-198, Miltenyi Biotec, NL). hPDCs were dispersed using TripLE (Life Technologies), suspended in a flow cytometry staining buffer solution (eBioscience Inc.,USA, Lot#E00015-1639), and stained in accordance to manufacturer’s instructions. In brief, 100 μl of cell suspension (up to 1 × 106) was mixed with 10 μl of MSC Phenotyping Cocktail and incubated for 10 minutes without light at 4 °C. Subsequently, hPDCs were washed and analyzed using BD FACS CantoTM using the cell analyzer (BD Biosciences, San Jose, CA) and FlowJo V10 software.
Leijten J., Teixeira L.S., Bolander J., Ji W., Vanspauwen B., Lammertyn J., Schrooten J, & Luyten F.P. (2016). Bioinspired seeding of biomaterials using three dimensional microtissues induces chondrogenic stem cell differentiation and cartilage formation under growth factor free conditions. Scientific Reports, 6, 36011.
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4

Cellular Affinity Determination Protocols

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Indirect and direct methods were used to test the cellular affinity according to previous protocols.[19] Briefly, for indirect method, lymphoma cells were incubated with dara (5 × 10−9m) and then a goat anti‐human secondary antibody (3 µg mL−1, ThermoFisher Scientific) in Flow Cytometry Staining Buffer Solution (eBioscience) at the concentration of 1 × 106 cells mL−1. For direct method,
N‐Hydroxysuccinimide –fluorescein (NHS–FITC, ThermoFisher)‐conjugated antibody was prepared and purified.[25] Then, FITC‐conjugated DTPA– or Df–dara were tested and processed using a LSRFortessa cell analyzer (BD Biosciences) and FlowJo software (Tree Star) for the mean fluorescence intensities.
Kang L., Li C., Rosenkrans Z.T., Huo N., Chen Z., Ehlerding E.B., Huo Y., Ferreira C.A., Barnhart T.E., Engle J.W., Wang R., Jiang D., Xu X, & Cai W. (2021). CD38‐Targeted Theranostics of Lymphoma with 89Zr/177Lu‐Labeled Daratumumab. Advanced Science, 8(10), 2001879.
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5

Daratumumab Binding Assay by Flow Cytometry

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Cells were washed with cold PBS buffer and incubated with Flow Cytometry Staining Buffer Solution (eBioscience, USA) at a concentration of 1 × 106 cells/mL at RT for 1 h. Cells were then incubated with 200 µL of daratumumab (5 or 25 µg/mL in abovementioned buffer) on ice for 60 min and then washed with cold PBS for three times. Cells were incubated with a goat anti-human secondary antibody (3 µg/mL, ThermoFisher Scientific) on ice for 30 min and washed again. The cells were analyzed using a LSRFortessa cell analyzer (BD Biosciences) and mean fluorescence intensities were processed using the FlowJo software (Tree Star, USA).
Kang L., Jiang D., England C.G., Barnhart T.E., Yu B., Rosenkrans Z.T., Wang R., Engle J.W., Xu X., Huang P, & Cai W. (2018). ImmunoPET imaging of CD38 in murine lymphoma models using 89Zr-labeled daratumumab. European journal of nuclear medicine and molecular imaging, 45(8), 1372-1381.
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