Pre sort buffer

Manufactured by BD

Sourced in United States

Pre-Sort buffer is a laboratory reagent used to prepare samples for flow cytometry analysis. It is designed to maintain the viability and integrity of cells or particles in the sample during the sorting process. The buffer helps to ensure consistent and accurate results by preventing cell clumping and optimizing the flow of the sample through the cytometer.

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Lab products found in correlation

7 aad viability dye, by Thermo Fisher Scientific (5 mentions) Fcr blocking reagent, by Miltenyi Biotec (4 mentions) Facsaria 3 sorp cell sorter, by BD (4 mentions) Facsdiva software, by BD (4 mentions) Cd3 allophycocyanin cy7 clone sk7, by BD (3 mentions) Cd4 phycoerythrin clone rpa t4, by BD (2 mentions) Cd8 fluorescein isothiocyanate clone rpa t8, by BD (2 mentions) Cd33 allophycocyanin clone wm53, by BD (2 mentions) Facsaria 3 sorp cell sorter on facsdiva software, by BD (2 mentions) Celltrace cfse dye, by Thermo Fisher Scientific (2 mentions) Dpbs ca2 mg2, by Thermo Fisher Scientific (2 mentions) Sh800s cell sorter, by Sony (2 mentions) Efluor 450, by Thermo Fisher Scientific (1 mentions) Cd133 2 293c3 pe antibody, by Miltenyi Biotec (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by R&D Systems (1 mentions) Anti cd44, by Thermo Fisher Scientific (1 mentions) Anti epcam, by Thermo Fisher Scientific (1 mentions) Aria 2 flow cytometer, by BD (1 mentions) Cd4 pe clone rpa t4, by BD (1 mentions) Cd3 apc cy7 clone sk7, by BD (1 mentions)

Close spelling variants of this product

BD FACS Pre-Sort Buffer

15 protocols using pre sort buffer

Fluorescence-Activated Cell Sorting of Immune Cell Subsets

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Cells were stained with cell-surface antibodies against CD3-allophycocyanin-Cy7 (clone SK7, BD Pharmingen, San Jose, USA), CD4-phycoerythrin (clone RPA-T4, BD Pharmingen), CD8-fluorescein isothiocyanate (clone RPA-T8; BD Pharmingen) and CD33-allophycocyanin (clone WM53, BD Pharmingen). Cells were washed twice with flow cytometry staining buffer and re-suspended in Pre-Sort buffer (BD Biosciences). 7-AAD viability dye (eBioscience, San Diego, USA) was used to gate live cells. BD FACSAria III SORP cell sorter on BD FACSDiva software (BD Biosciences) was used for sorting pure CD4⁺ (7AAD^–CD3⁺CD4⁺CD8^–CD33^–), CD8⁺ (7AAD^–CD3⁺CD4^–CD8⁺CD33^–) and CD33⁺(7AAD^–CD3^–CD4^–CD8^–CD33⁺) populations. Applicable measures were taken to ensure minimal sorter-induced cell stress (SICS). Data analyses were performed on FlowJo V10 software (FlowJo, Ashland, USA).

Sasidharan Nair V., Saleh R., Taha R.Z., Toor S.M., Murshed K., Ahmed A.A., Kurer M.A., Abu Nada M., Al Ejeh F, & Elkord E. (2020). Differential gene expression of tumor-infiltrating CD4+ T cells in advanced versus early stage colorectal cancer and identification of a gene signature of poor prognosis. Oncoimmunology, 9(1), 1825178.

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Quantifying Adipose Precursor Cells by Flow Cytometry

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The fluorescent signal generated by Click-iT^® EdU labeling was detected using logarithmic amplification of 633/635 nm excitation and a red emission filter; 50,000 events were counted using a low flow rate during acquisition. For adipose precursor cells sorting, SVF were incubated after EdU labeling, with CD34 (eFluor 450, eBioscience 48-0341-82, 1:20), CD31 (Alexa Fluor 488, BD 563607, 1:20), and CD45 (BUV395, BD 565967, 1:100) for 1 h in pre-sort buffer (BD, 563503) on ice and protected from light. Raw data were processed using FlowJo software (Tree Star, Ashland, OR, USA).

Ribas-Latre A., Santos R.B., Fekry B., Tamim Y.M., Shivshankar S., Mohamed A.M., Baumgartner C., Kwok C., Gebhardt C., Rivera A., Gao Z., Sun K., Heiker J.T., Snyder B.E., Kolonin M.G, & Eckel-Mahan K.L. (2021). Cellular and physiological circadian mechanisms drive diurnal cell proliferation and expansion of white adipose tissue. Nature Communications, 12, 3482.

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CD133+ Cell Enrichment under Hypoxia

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Cells in the logarithmic growth phase were cultured under hypoxic conditions or normal conditions for 24 h at 37°C. They were subsequently washed three times with PBS, collected by centrifugation at 300 × g for 5 min at room temperature, and resuspended and dissociated into single cell suspension in the Pre-Sort Buffer (BD FACS^™) by repeatedly pipetting. The cells were then centrifuged at 300 × g for 5 min at room temperature and the supernatant was discarded. The cells were washed with 8 ml PBS, blocked with 2 ml FcR Blocking Reagent (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for 10 min at 4°C and incubated with 2 ml CD133/2 (293C3)-PE antibodies (dilution, 1:1,000; cat. no. 130-090-853; Miltenyi Biotec GmbH) for 10 min at 4°C in the dark. The cells were washed once with 2 ml PBS and then the proportion of CD133+ cells were detected using the BD FACSCalibur^™ analyzer with FlowJo 7.6 software.

Zeng F., Chen H., Zhang Z., Yao T., Wang G., Zeng Q., Duan S, & Zhan Y. (2018). Regulating glioma stem cells by hypoxia through the Notch1 and Oct3/4 signaling pathway. Oncology Letters, 16(5), 6315-6322.

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Senescence Sorting by CellTrace CFSE

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After senescence induction, the cells were washed with DPBS+Ca²⁺/Mg²⁺(Gibco) and incubated with 5 µM CellTrace CFSE dye (Thermo Fisher) diluted in DPBS+Ca²⁺/Mg²⁺ for 20 min in the incubator. Afterwards, the staining solution was removed, and the cells were washed with culture medium and cultured in fresh medium for 10 (senescent cells) or 4 (control cells) days in the incubator. After CellTrace CFSE staining, the cells were collected, centrifuged, resuspended in pre-sort buffer (BD Biosciences, Franklin Lakes, NJ, USA, cat. number: 563503) at a concentration of 1 × 10⁶ cells/mL and sorted using the Sony SH800S Cell Sorter. The gates were adjusted to separate senescent and senescence-escaped cells by using control and senescent cells stained with CellTrace CFSE.

Tóth F., Moftakhar Z., Sotgia F, & Lisanti M.P. (2024). In Vitro Investigation of Therapy-Induced Senescence and Senescence Escape in Breast Cancer Cells Using Novel Flow Cytometry-Based Methods. Cells, 13(10), 841.

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Fluorescent-Activated Cell Sorting Protocol

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Single-cell suspensions were incubated with 0.05 μg of anti-CD44 (#17-0441-82, Thermo Fisher Scientific, MA), 0.05 μg of anti-EpCAM (#14-9326-95, Thermo Fisher Scientific, MA), and DAPI (R&D Systems, MN). Cells were then washed and resuspended in presort buffer (BD Biosciences, CA) for fluorescence-activated cell sorting (FACS) using the BD Biosystems Aria II flow cytometer. For FP-expressing TF reporter cells, single cells were suspended in media and sorted using 640-nm (Zombie NIR, 750LP filter), 355-nm (Calcein Blue AM, 460/50 filter), 488-nm (CD45-PerCP, 692/40 filter), and 488-nm (GFP, 530/40 filter) lasers. Side scatter width versus forward scatter (FSC) area and trigger pulse width versus FSC criteria were used to discriminate doublets and gate only single cells. We sorted viable FP-positive and FP-negative single cells into round-bottom 5-ml FACS tubes. Cells were snap-frozen immediately after sorting and stored at −80°C before whole transcriptome amplification, library preparation, and sequencing.

Gopal P., Petty A., Rogacki K., Bera T., Bareja R., Peacock C.D, & Abazeed M.E. (2022). Multivalent state transitions shape the intratumoral composition of small cell lung carcinoma. Science Advances, 8(50), eabp8674.

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Multiparameter flow cytometry immune cell sorting

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Following phosphate-buffered saline (PBS) washing, single cell suspension from tumor tissue was washed and resuspended in 100 µL of flow cytometry staining buffer (PBS with 1% fetal calf serum (FCS) and 0.1% sodium azide). Fc receptor (FcR) Blocking Reagent, human (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to block FcR. Cells were stained with cell surface antibodies against CD3-APC-Cy7 (clone SK7, BD Pharmingen, San Jose, USA), CD4-PE (clone RPA-T4, BD Pharmingen), CD8-FITC (clone RPA-T8; BD Pharmingen), CD33-APC (clone WM53, BD Pharmingen) and 7-AAD viability dye (eBioscience, San Diego, USA) to exclude dead cells and gate on live cells.
Cells were washed with flow cytometry staining buffer then re-suspended in Pre-Sort buffer (BD Biosciences). BD FACSAria III SORP cell sorter on BD FACSDiva software (BD Biosciences) was used for sorting pure CD8⁺ (7AAD^–CD3⁺CD4^–CD8⁺CD33^–), CD4⁺ (7AAD^–CD3⁺CD4⁺CD8^–CD33^–) and CD33⁺(7AAD^–CD3^–CD4^–CD8^–CD33⁺) populations. The sorting strategy is shown in figure 1A. We used stringent gating strategy and applicable measures to ensure minimal sorter-induced cell stress. High purities of sorted immune cell populations were always checked and confirmed. FlowJo V.10 software (FlowJo, Ashland, USA) was used for data analyzes.

Saleh R., Sasidharan Nair V., Toor S.M., Taha R.Z., Murshed K., Al-Dhaheri M., Khawar M., Petkar M.A., Abu Nada M., Al-Ejeh F, & Elkord E. (2020). Differential gene expression of tumor-infiltrating CD8+ T cells in advanced versus early-stage colorectal cancer and identification of a gene signature of poor prognosis. Journal for Immunotherapy of Cancer, 8(2), e001294.

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Multicolor Flow Cytometry Analysis

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Cells isolated from tissues were washed with PBS and re-suspended in 100 μl flow cytometry staining buffer (PBS with 1% FCS and 0.1% sodium azide). Fc receptors (FcR) were first blocked using FcR Blocker (Miltenyi Biotech, Bergisch Gladbach, Germany). 7-AAD viability dye (eBioscience, San Diego, USA) was used to gate live cells. Cells were then stained with cell surface antibodies against CD33-Fluorescein isothiocyanate (clone HIM3-4; BD Biosciences, Oxford, UK), CD14-phycoerythrin-Cy7 (clone M5E2; BD Biosciences), CD15-allophycocyanin (clone HI98; BioLegend, San Diego, USA), HLA DR-phycoerythrin (clone G46-6; BD Biosciences) or CD11a-phycoerythrin (clone G43-25B; BD Pharmingen, San Jose, USA) and incubated at 4 °C for 30 min. Cells were then washed twice with flow cytometry staining buffer and data were acquired by BD LSRFortessa X-20 flow cytometer (BD Biosciences).
For sorting, cells were re-suspended in Pre-Sort buffer (BD Biosciences). BD FACSAria III SORP cell sorter with BD FACSDiva software (BD Biosciences) was used. Applicable measures were taken to ensure minimal sorter-induced cell stress (SICS). Data analyses were performed on FlowJo V10 software (FlowJo, Ashland, USA).

Sasidharan Nair V., Saleh R., Toor S.M., Taha R.Z., Ahmed A.A., Kurer M.A., Murshed K., Alajez N.M., Abu Nada M, & Elkord E. (2020). Transcriptomic profiling disclosed the role of DNA methylation and histone modifications in tumor-infiltrating myeloid-derived suppressor cell subsets in colorectal cancer. Clinical Epigenetics, 12, 13.

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Isolation of CD4+ and CD8+ TILs

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Single cell suspensions from TT specimens were resuspended in 100 µL of flow cytometry staining buffer (PBS with 1% FCS and 0.1% sodium azide). An FcR Blocking Reagent (Miltenyi Biotec) was used to block Fc receptors (FcR) and a 7-AAD viability dye (eBioscience, San Diego, CA, USA) was used to gate live cells. Cells were stained with surface antibodies against CD3-allophycocyanin-Cy7 (clone SK7, BD Pharmingen, San Jose, CA, USA), CD4-phycoerythrin (clone RPA-T4, BD Pharmingen), and CD8-fluorescein isothiocyanate (clone RPA-T8; BD Pharmingen). Cells were washed twice with flow cytometry staining buffer prior to re-suspension in Pre-Sort buffer (BD Biosciences, Oxford, UK). A BD FACSAria III SORP cell sorter on BD FACSDiva software (BD Biosciences) was used for sorting pure CD4⁺ (7AAD⁻CD3⁺CD4⁺CD8⁻), and CD8⁺ (7AAD⁻CD3⁺CD4⁻CD8⁺) TILs. Relevant procedures were followed to ensure minimum sorter-induced cell stress (SICS), as previously described [18 (link)]. Flow cytometric analyses were performed on FlowJo V10 software (FlowJo, Ashland, OH, USA).

Toor S.M., Sasidharan Nair V., Saleh R., Taha R.Z., Murshed K., Al-Dhaheri M., Khawar M., Ahmed A.A., Kurer M.A., Abu Nada M, & Elkord E. (2021). Transcriptome of Tumor-Infiltrating T Cells in Colorectal Cancer Patients Uncovered a Unique Gene Signature in CD4+ T Cells Associated with Poor Disease-Specific Survival. Vaccines, 9(4), 334.

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Isolation of Amniotic Fluid Neutrophils

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Amniotic fluid samples were passed through a sterile 15-μm filter (Cat# 43-50015-03; pluriSelect Life Science, Leipzig, Germany) to remove epithelial cells and centrifuged at 200 × g for 5 minutes at room temperature (n=16). The cell pellet (mostly leukocytes⁴⁶) was washed with 1× phosphate-buffered saline (1× PBS; Life Technologies, Grand Island, NY), resuspended at 1×10⁶ cells in 100μL of BD FACS stain buffer (Cat#554656; BD Biosciences, San Jose, CA) containing 20% human FcR blocking reagent (Miltenyi Biotec, San Diego, CA, USA), and incubated for 10 min at 4°C. Next, amniotic fluid cells were incubated with the following extracellular fluorochrome-conjugated anti-human antibodies (BD Biosciences) for 30 min at 4°C in the dark: CD15-FITC (fluorescein isothiocyanate) (clone W6D3, Cat#562370) and CD14-APC-Cy7™ (clone MφP9, Cat#557831). After a washing with 1× PBS, the cells were resuspended in pre-sort buffer (Cat# 563503, BD Biosciences) at a concentration of 5×10⁶ cells/mL. Amniotic fluid neutrophils (CD15+CD14- cells) were purified using a BD FACSAria cell sorter (BD Biosciences) and BD FACSDiva 6.0 software (BD Biosciences). The purity of amniotic fluid neutrophils ranged from 92% to 99% (Figure 1). The purified neutrophils were then resuspended in RLT buffer (Qiagen, Germantown, MD) and stored at -80°C until use.

Gomez-Lopez N., Romero R., Xu Y., Leng Y., Garcia-Flores V., Miller D., Jacques S.M., Hassan S.S., Faro J., Alsamsam A., Alhousseini A., Gomez-Roberts H., Panaitescu B., Yeo L, & Maymon E. (2017). Are Amniotic Fluid Neutrophils in Women with Intra-Amniotic Infection and/or Inflammation of Fetal or Maternal Origin?. American journal of obstetrics and gynecology, 217(6), 693.e1-693.e16.

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Sorting and Characterization of Neuronal Cell Populations

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Fibroblast cells were detached by Accutase (Millipore) and resuspended in Pre‐Sort buffer (BD Biosciences). Red Fluorescent Protein (RFP) positive cells were aseptically sorted in a FACS ARIA Fusion machine (BD Biosciences) using a 130 μm nozzle at 20 psi. Cells were sorted in a 6‐well plate in prewarmed fibroblast growth medium, 2,000 RFP positive cells per well. Determination of RFP negative and RFP positive populations, after doublet discrimination, was based on gating of unelectroporated and electroporated cells in 616/23 and 695/40 filters.
Estimation of populations of forebrain and midbrain cells was done by flow cytometry on BD FACS Aria Fusion. Mature neurons were prepared for FACS as described, stained with DAPI and labeled with a TUJ1 antibody coupled to Alexa488 (TUJ1‐Alexa488). Forebrain cells we also labeled with MAP2‐Alexa647, and midbrain cells were labeled with TH‐Alexa647. Population of single cells was identified by doublet discrimination followed by DAPI positive gating. Neuronal and non‐neuronal populations were identified by gating of TUJ1‐Alexa 488 positive and negative populations of cells, respectively. MAP2+ and TH+ populations were identified by gating from TUJ1+ population.

Bell S., Peng H., Crapper L., Kolobova I., Maussion G., Vasuta C., Yerko V., Wong T.P, & Ernst C. (2016). A Rapid Pipeline to Model Rare Neurodevelopmental Disorders with Simultaneous CRISPR/Cas9 Gene Editing. Stem Cells Translational Medicine, 6(3), 886-896.

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