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Prosort software

Manufactured by Bio-Rad
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ProSort software is a data analysis tool designed for flow cytometry applications. It provides users with the ability to analyze and sort cell populations based on multiple parameters. The software offers features for customizing data visualization and gating, allowing users to efficiently process and interpret flow cytometry data.

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Lab products found in correlation

S3e cell sorter, by Bio-Rad (3 mentions) S3 flow cytometer, by Bio-Rad (2 mentions) S3 cell sorter, by Bio-Rad (2 mentions) Anti mouse sca 1 antibody conjugated with af488, by Thermo Fisher Scientific (2 mentions) Aria 2 sorp cell sorter, by BD (1 mentions) Mitotracker red cm h2xros, by Thermo Fisher Scientific (1 mentions) Phenol red free dmem, by Corning (1 mentions) Rnase a, by Merck Group (1 mentions) Trueline, by Nippon Genetics (1 mentions) Annexin 5 fitc conjugate, by Thermo Fisher Scientific (1 mentions) Labtek permanox chamber slides, by Thermo Fisher Scientific (1 mentions) Tryple select, by Thermo Fisher Scientific (1 mentions) Red blood lysis solution, by Miltenyi Biotec (1 mentions) Dm rb leica microscope, by Leica camera (1 mentions) Las image acquisition software, by Leica camera (1 mentions) Automacs running buffer, by Miltenyi Biotec (1 mentions)

9 protocols using prosort software

1

Flow Cytometry Analysis and Sorting

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Quantitative analysis by flow cytometry was performed using a Propel Laboratories Avalon cytometer with a 100 μm nozzle and standard GFP/RFP filter sets. Data were further analyzed using BioRad ProSort software.
Fluorescence-activated cell sorting (FACS) was performed using a BD AriaII SORP cell sorter with a 100 μm nozzle at Dana-Farber flow cytometry core facility.
Guo Y., VanDusen N.J., Zhang L., Gu W., Sethi I., Guatimosim S., Ma Q., Jardin B.D., Ai Y., Zhang D., Chen B., Guo A., Yuan G.C., Song L.S, & Pu W.T. (2017). Analysis of Cardiac Myocyte Maturation Using CASAAV, A Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo. Circulation research, 120(12), 1874-1888.
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2

Mitochondrial Membrane Potential Assay

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MO3.13 cells were seeded in 24 well plates (25000 cells/cm2) and treated as described before; after treatments, the cells were stained with MitoTracker Red CM-H2XRos (Molecular Probes-Life Technologies, Carlsbad, CA, USA) for 30 min at 37 °C in humidified atmosphere containing 5% CO2. MitoTracker Red CM-H2XRos was diluted with anhydrous DMSO to obtain 1 mM of stock solution and used 1 μM as final concentration. After staining, cells were harvested and spun for 5 min at 1400 rpm; the obtained pellets were washed with PBS and spun again for 5 min at 1400 rpm. Flow cytometry was performed by using a S3 flow cytometer (Bio-Rad) equipped with a 488 nm and 561 diode-pumped solid-state lasers. MitoTracker Red CM-H2XRos was excited with a 561 nm laser and fluorescence emission was collected through a 605/40 nm band-pass filter and 570 nm long-pass fiter. At least 20 000 gated events were acquired for each sample. Data were analyzed using the Bio-Rad ProSort Software.
Voccoli V., Tonazzini I., Signore G., Caleo M, & Cecchini M. (2014). Role of extracellular calcium and mitochondrial oxygen species in psychosine-induced oligodendrocyte cell death. Cell Death & Disease, 5(11), e1529-.
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3

Quantification of Cellular Uptake of eGFP-CPP

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Cells were seeded at a density of 1 × 105 cells per well in a 24-well plate and grown at 37 °C in a 5% CO2 atmosphere in DMEM supplemented with 10% (v/v) FBS for 24 h. Cells were then incubated with 10 μM of the designated eGFP-CPP fusion protein in phenol red free DMEM (Life Technologies, #21063-029) at 37 °C for 1 h, followed by washing three times with heparin sulphate and twice with phenol red free DMEM for two times before addition of trypsin (50 µl/well; Corning, #25-052-CI). Trypsinised cells were diluted with PBS (0.45 mL) and analysed on a Bio-Rad S3e Cell Sorter for minimum 10,000 events per sample. Data were analysed on ProSort software (Bio-Rad), and geometric mean values of green fluorescence (526 nm) of live cells in each sample from excitation with a 488-nm laser were taken for quantification.
Patel S.G., Sayers E.J., He L., Narayan R., Williams T.L., Mills E.M., Allemann R.K., Luk L.Y., Jones A.T, & Tsai Y.H. (2019). Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines. Scientific Reports, 9, 6298.
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4

Cell Cycle Analysis of HeLa Cells

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HeLa cells were seeded at the density of 2.5 × 105 cells in 4 ml culture medium in a 25 cm2 flask (Trueline, Nippon
Genetics Co., Ltd.) and cultured with 100 µl of filter sterilized bacterial sonic cell lysate at 37°C under 5% CO2 in air. After 24
hr incubation, cells were collected by centrifugation at 1,000 ×g at 4°C for 5 min and fixed with 70% ethanol on ice for 2 hr. The cells were
then collected and treated with 0.1% Triton X-100 in PBS at room temperature for 15 min. Subsequently, cells were collected, suspended in PBS containing 0.25
mg/ml of RNase A (Sigma-Aldrich, St. Louis, MO, U.S.A.) and incubated at 37°C for 15 min and further stained with propidium iodide (PI; 50
µg/ml) at 4°C for 30 min under the dark condition. Flow cytometry analysis of DNA content of the cells was performed with a
S3 cell sorter (Bio-Rad Laboratories Inc., Hercules, CA, U.S.A.), and 10,000 events were collected. Cell cycle analysis was performed by ProSort software
(Bio-Rad Laboratories Inc.).
SOMROOP S., HATANAKA N., AWASTHI S.P., OKUNO K., ASAKURA M., HINENOYA A, & YAMASAKI S. (2017). Campylobacter upsaliensis isolated from dogs produces high titer of cytolethal distending toxin. The Journal of Veterinary Medical Science, 79(3), 683-691.
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5

Annexin V-FITC and PI Apoptosis Assay

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After treatments, MO3.13 cells were harvested and spun for 5 min at 1400 rpm; the obtained pellets were washed with PBS 1 × and spun again for 5 min at 1400 rpm. Cells were re-suspended in binding buffer (10 mmol/l HEPES, 135 mmol/l NaCl, 5 mmol/l CaCl2) containing Annexin V-FITC conjugate 1 μM (Invitrogen-Life Technologies, Carlsbad, CA, USA) and PI 100 nM (Sigma-Aldrich, St. Louis, MO, USA) and incubated in ice for 30 min. Flow cytometry was performed by using a S3 flow cytometer (Bio-Rad, Hercules, CA, USA) equipped with 488 and 561 nm diode-pumped solid-state lasers. Annexin V-FITC was excited using the 488 nm laser, and fluorescence was collected through a 552/50 nm band-pass filter and 505 nm long-pass filter; PI was excited with the 561 nm laser and the fluorescence was collected through a 605/40 nm band-pass filter and a 570 nm long-pass fiter. At least 20 000 gated events were acquired for each sample. Data were analysed using the Bio-Rad ProSort software.
Voccoli V., Tonazzini I., Signore G., Caleo M, & Cecchini M. (2014). Role of extracellular calcium and mitochondrial oxygen species in psychosine-induced oligodendrocyte cell death. Cell Death & Disease, 5(11), e1529-.
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6

Isolation and Characterization of Vascular Progenitor Cells from Bone Marrow Mononuclear Cells

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hBM-MNCs were stained with anti-CD18 PE, anti-CD31 PE-Cy7, and a cocktail of FITC-conjugated antibodies, including anti-CD3, anti-CD20, anti-CD14, and anti-CD66abce. After 30 min at 4°C cells were washed with prerefrigerated autoMACS Running Buffer and processed for cell sorting on S3™ Cell Sorter (Bio-Rad, Hercules, CA) equipped with ProSort® Software (Bio-Rad) applying “Purity” sort mode. Sorted cells were then cytocentrifuged on glass slides to perform May Grünwald–Giemsa and cytochemical staining or alternatively plated in DMEM/10%PhABS using TC 24-well plates. After 5 days of culture, cells were exposed to mesengenic and angiogenic differentiating conditions, tested for acetylated low-density lipoprotein (Ac-LDL) uptake and phagocytosis, or detached by TrypLE Select® (Life Technologies) digestion, and analyzed by flow cytometry, as described below.
Nestin detection was performed on sorted cells seeded in two-well Lab-Tek Permanox® Chamber Slides (Thermo Fisher Scientific, Waltham, MA). Fluorescence-activated cell sorting (FACS) was extended to hematopoietic lineage antigens using a cocktail of CD64-FITC, CD31-PE/Cy7, and CD14-PE antibodies. Within CD64brightCD31bright population, CD14neg and CD14+ cells were separated on S3 Cell Sorter and processed as described above.
Pacini S., Barachini S., Montali M., Carnicelli V., Fazzi R., Parchi P, & Petrini M. (2016). Mesangiogenic Progenitor Cells Derived from One Novel CD64brightCD31brightCD14neg Population in Human Adult Bone Marrow. Stem Cells and Development, 25(9), 661-673.
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7

Isolation and Characterization of Sca-1+ Muscle Stem Cells

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Single cell suspension was prepared from hindlimb skeletal muscle as discussed above (all the procedures were performed at 4°C unless otherwise specified). The single cell suspension containing 5.0 million cells were stained using anti-mouse Sca-1 antibody conjugated with AF488 (Thermo Fisher Scientific) and incubated for 1h. The cells were washed twice with staining buffer and resuspended in 1 mL of staining buffer. The cells were sorted based on the selection of negative and positive sort mode using Bio-Rad S3e cell sorter (Bio Rad). The results were analyzed using Prosort software (Bio Rad). The positive and negative populations were collected in muscle derived stem cell (MDSC) media. Gatings were carried out using unstained and FMO controls in which 0.2 X 10 6 cells each were taken respectively.
The sorted Sca-1 + cells were divided into two sets; the first set labeled as the ex vivo Sca-1 + sample from which the protein was isolated by addition of lysis buffer to the cell pellet. The second set of Sca-1 + cells were cultured in MDSC media for their expansion which represent the in vitro sample.
Kapoor S., Subba P., Shenoy P S, & Bose B. (2021). Sca1+ Progenitor Cells (Ex vivo) Exhibits Differential Proteomic Signatures From the Culture Adapted Sca1+ Cells (In vitro), Both Isolated From Murine Skeletal Muscle Tissue. Stem cell reviews and reports, 17(5).
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8

Isolation and Characterization of Sca-1+ Muscle Stem Cells

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Single cell suspension was prepared from hindlimb skeletal muscle as discussed above (all the procedures were performed at 4°C unless otherwise specified). The single cell suspension containing 5.0 million cells were stained using anti-mouse Sca-1 antibody conjugated with AF488 (Thermo Fisher Scientific) and incubated for 1h. The cells were washed twice with staining buffer and resuspended in 1 mL of staining buffer. The cells were sorted based on the selection of negative and positive sort mode using Bio-Rad S3e cell sorter (Bio Rad). The results were analyzed using Prosort software (Bio Rad). The positive and negative populations were collected in muscle derived stem cell (MDSC) media. Gatings were carried out using unstained and FMO controls in which 0.2 X 10 6 cells each were taken respectively.
The sorted Sca-1 + cells were divided into two sets; the first set labeled as the ex vivo Sca-1 + sample from which the protein was isolated by addition of lysis buffer to the cell pellet. The second set of Sca-1 + cells were cultured in MDSC media for their expansion which represent the in vitro sample.
Kapoor S., Subba P., P S.S., & Bose B. (2020). Sca1+ cells as direct isolate (ex vivo) versus in vitro cultured exhibit differential proteomic signatures in murine skeletal muscle.
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9

Sorting and Characterizing Eosinophils by CD157 and FLAER

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To demonstrate that the CD157 -/FLAER + granulocytic population consisted of eosinophils, we performed sorting studies by means of a S3 Cell Sorter (Bio-Rad, Hercules, USA-CA) equipped with ProSort ® Software (Bio-Rad) and applying "Purity" sort mode. 500 µL of peripheral blood, collected in K3 EDTA pre-treated tube, were stained with FLAER-Alexa Fluor 488 and CD157-PE reagents. After 30 sec at 4℃, erythrocytes were lysed applying Red Blood Lysis Solution from Miltenyi Biotec (Bergisch Gladbach, Germany) and washed with pre-refrigerated AutoMACS Running Buffer (Miltenyi Biotec). Gating granulocytic events as FSC high SSC high , sort regions were defined on PE vs FITC cytogram in order to collect CD157 + /FLAER + and CD157 -/FLAER -frac-tions. About 250,000 sorted cells were then cytocentrifuged on glass slides and processed for May Grünwald-Giemsa stain on a Wescor Aerospray ® 7120 automatic slide stainer (ELITech Group, Puteaux, France). Pictures were taken using standard DM RB Leica microscope (Leica, Wetzlar, Germany) equipped with LAS image acquisition software (Leica).
Carulli G., Marini A., Sammuri P., Domenichini C., Ottaviano V., Pacini S, & Petrini M. (2015). Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry. Journal of clinical and experimental hematopathology : JCEH, 55(2).
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