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Guava easycyte mini flow cytometry system

Manufactured by Merck Group
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The Guava EasyCyte Mini Flow Cytometry System is a compact, automated flow cytometer designed for cell analysis. It utilizes laser-based technology to rapidly detect and analyze individual cells within a sample. The system provides quantitative data on various cell properties, including size, granularity, and fluorescence intensity.

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Lab products found in correlation

Guava acquisition and analysis software, by Merck Group (5 mentions) Ribonuclease, by Abcam (2 mentions) Nocodazole, by Abcam (2 mentions) Acridine orange, by Merck Group (2 mentions) Cyto id autophagy detection kit, by Enzo Life Sciences (2 mentions) Ethanol, by Labsynth (1 mentions) Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions) Dichloromethane, by Labsynth (1 mentions) Guava cell cycle reagent, by Merck Group (1 mentions) Crystal violet, by Merck Group (1 mentions) Guava nexin reagent, by Merck Group (1 mentions) Penicillin streptomycin, by Vitrocell (1 mentions) Piroxicam, by Pfizer (1 mentions) Sytox, by Thermo Fisher Scientific (1 mentions) Mid 15b4, by Enzo Life Sciences (1 mentions)

8 protocols using guava easycyte mini flow cytometry system

1

Anti-inflammatory Activity Evaluation

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All solvents and chemicals were purchased from commercial sources and were used without further purification. Dichloromethane and ethanol (analytical grade) were obtained from Labsynth (Diadema, SP, Brazil). Trichloroacetic acid, Tween 80®, dexamethasone (purity > 90%), dimethylsulfoxide (DMSO), carrageenan, croton oil and hexadecyltrimethylammonium bromide buffer (HTAB) were obtained from Sigma-Aldrich Laboratories (San Loius, MO, USA). Piroxicam was obtained from Pfizer (New York, NY, USA). Culture medium RPMI 1640 and fetal bovine serum (SBF) were from Gibco (Waltham, MA, USA). Penicillin/streptomycin (1000 U/mL:1000 mg/mL) and trypsin-EDTA 0.25% were acquired from Vitrocell (Campinas, SP, Brazil). Doxorubicin (chlorohydrate 50 mg; purity > 90%) was obtained from Eurofarma (São Paulo, SP, Brazil). Crystal violet, Guava Nexin Reagent, Guava Multicaspase kit (Caspase Reagent Working Solution, Apoptosis Wash Buffer and Caspase 7-AAD Reagent Working Solution) and Guava Cell Cycle Reagent were acquired from Merck-Millipore (Darmstadt, Germany). Flow cytometry experiments and analysis were performed using a Guava EasyCyte Mini Flow Cytometry System (Millipore). Plethysmometer apparatus (7140) was purchased from Ugo Basile (Gemonio, VA, Italy) and a grinder (Disperser T 10 basic) from IKA (Campinas, SP, Brasil).
de Paiva P.P., Nunes J.H., Nonato F.R., Ruiz A.L., Zafred R.R., Sousa I.M., Okubo M.Y., Kawano D.F., Monteiro P.A., Foglio M.A, & Carvalho J.E. (2020). In Silico, In Vitro, and In Vivo Antitumor and Anti-Inflammatory Evaluation of a Standardized Alkaloid-Enriched Fraction Obtained from Boehmeria caudata Sw. Aerial Parts. Molecules, 25(17), 4018.
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2

Evaluating Cytotoxicity of CPT Derivatives

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Human glioma U1242 cells (1×106) were seeded in a 100-mm cell culture dish and cultured for 1 day to allow cell attachment. The cells were treated with CPT at the concentration of 2×IC50free, conjugated CPT at the concentration of 2×IC50cojugated and PPA-G4.5-PEG conjugates at the equivalent concentration of CPT-G4.5-PEG conjugates for various lengths of time (6, 12, and 24 h). The cells treated with PBS were used as a control. At the end of each treatment, the cells were washed with PBS and re-suspended in fresh cell culture medium following trypsinization. Following the centrifugal removal of the medium, the cells were fixed with cold 70% ethanol and maintained at 4°C for 1 h. Finally, the cells were washed with PBS three times and incubated with RNase at a final concentration of 1 μg/mL and propidium iodide at a final concentration of 50 μg/mL at 37 °C for 30 min. The cells were then immediately analyzed by flow cytometry using a Guava EasyCyte mini flow cytometry system (Millipore, Billerica, MA).27 (link)
Zolotarskaya O.Y., Xu L., Valerie K, & Yang H. (2015). Click synthesis of a polyamidoamine dendrimer-based camptothecin prodrug. RSC advances, 5(72), 58600-58608.
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3

Cell Cycle Analysis of CD38 CAMs

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The CAMs were synchronized in G2/M phase by treating the cell with nocodazole (100 ng/ml, Abcam) for 12 h [18 (link)]. After soft wash with cold PBS, the CD38+/+ or CD38−/− CAMs was incubated with vehicle (Vehl) or baf for 24 h and then collected by trypsinization. Furthermore, the CAMs was harvested by centrifugation on 700 rpm at 4°C for 10 min and washed twice with PBS. The cells were then fixed in ice-cold 70% ethanol for 30 min at 4℃ and washed twice with PBS. The cells were treated with ribonuclease (100 μg/ml, Abcam) followed by addition of 200 μL propidium iodide (PI, 50 μg/ml, Abcam). Samples were analyzed using a Guava Easycyte Mini Flow Cytometry System and the Guava acquisition and analysis software (Guava Technologies, Hayward, CA). The cell number in G0/G1, S or G2/M phase was divided by the total cell number (G0/G1+S+G2/M) to indicate the percentage of cells in specific phases.
Bao J., Li G., Yuan X., Gulbins E, & Li P. (2017). Contribution of p62 to Phenotype Transition of Coronary Arterial Myocytes with Defective Autophagy. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 41(2), 555-568.
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4

Cell Cycle Analysis of CD38 CAMs

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The CAMs were synchronized in G2/M phase by treating the cell with nocodazole (100 ng/ml, Abcam) for 12 h [18 (link)]. After soft wash with cold PBS, the CD38+/+ or CD38−/− CAMs was incubated with vehicle (Vehl) or baf for 24 h and then collected by trypsinization. Furthermore, the CAMs was harvested by centrifugation on 700 rpm at 4°C for 10 min and washed twice with PBS. The cells were then fixed in ice-cold 70% ethanol for 30 min at 4℃ and washed twice with PBS. The cells were treated with ribonuclease (100 μg/ml, Abcam) followed by addition of 200 μL propidium iodide (PI, 50 μg/ml, Abcam). Samples were analyzed using a Guava Easycyte Mini Flow Cytometry System and the Guava acquisition and analysis software (Guava Technologies, Hayward, CA). The cell number in G0/G1, S or G2/M phase was divided by the total cell number (G0/G1+S+G2/M) to indicate the percentage of cells in specific phases.
Bao J., Li G., Yuan X., Gulbins E, & Li P. (2017). Contribution of p62 to Phenotype Transition of Coronary Arterial Myocytes with Defective Autophagy. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 41(2), 555-568.
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5

Measuring Cell Injury via Flow Cytometry

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MVECs were harvested and washed with PBS and then PI (final concentration, 2μg/ml) was added during or after LCWE or saponin incubation. After 30 min, the cells were incubated with SYTOX (Life Technologies, Grand Island, NY, USA) at 37 °C for 4 hours in the presence of PI. Stained cells were run through a Guava Easycyte Mini Flow Cytometry System (Guava Technologies, Hayward, CA, USA) according to the manufacturer's instructions (32 (link)) and analyzed with Guava acquisition and analysis software (Guava Technologies). Cell populations with strong PI staining indicate cell injury.
Chen Y., Yuan M., Xia M., Wang L., Zhang Y, & Li P.L. (2016). Instant membrane resealing in nlrp3 inflammmasome activation of endothelial cells. Frontiers in bioscience (Landmark edition), 21, 635-650.
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6

Quantifying Autophagy and Lysosomal Activity

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The Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA) was used to detect AP 5 (link). Briefly, CAMs (1 × 105/ml) were collected and centrifuged (400 × g, 5 min.) at the end of the treatment. Then, CAMs were incubated with 0.5 ml of freshly diluted Cyto-ID Green Detection Reagent (1:4000) for 30 min. at 37°C in the dark. Without washing, stained CAMs were run in the green (FL1) channel with a Guava Easycyte Mini Flow Cytometry System (Guava Technologies, Hayward, CA, USA) and analysed with Guava acquisition and analysis software (Guava Technologies). The enhancement of Cyto-ID Green dye signal indicates an increase in AP.
In addition, acridine orange (Sigma-Aldrich, St. Louis, MO, USA) was used to detect APLs. CAMs (1 × 106/ml) were stained with acridine orange (1:5000) for 17 min. After washes, CAMs were harvested in phenol red-free growth medium. Green (510–530 nm) and red (>650 nm) fluorescence emission from 104 cells illuminated with blue (488 nm) excitation light was measured with Flow Cytometry System and analysed with Guava acquisition and analysis software. The mean red/green fluorescence ratio was calculated to indicate the change of intracellular APLs.
Xu M., Li X.X., Chen Y., Pitzer A.L., Zhang Y, & Li P.L. (2014). Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes. Journal of Cellular and Molecular Medicine, 18(11), 2165-2175.
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7

Annexin A5 and Ceramide Expression

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The surface expression of annexin A5 and ceramide in MAECs was analyzed by flow cytometry. In brief, MAECs were harvested and washed with PBS 0.2% Tween 20 and incubated with anti-annexin A5 antibody (1:200, Abcam, Cambridge, MA, United States) and anti-ceramide antibody (1:20, MID 15B4, Enzo, Ann Arbor, MI, United States) for 30 min on ice. The cells were fixed with paraformaldehyde, washed with PBS 0.2% Tween 20, and resuspended in ice cold PBS with 10% FCS and 1% sodium azide. Cells were analyzed through a Guava Easycyte Mini Flow Cytometry System (Guava Technologies, Hayward, CA, United States) using Guava acquisition and analysis software.
Chen Y., Li G., Bhat O.M., Li X., Zhang Y, & Li P.L. (2022). Impairment of Ceramide-Mediated Endothelial Instant Membrane Resealing During Diabetes Mellitus. Frontiers in Physiology, 13, 910339.
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8

Quantifying Autophagy Using Cyto-ID and Acridine Orange

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The Cyto-ID Autophagy Detection Kit (Enzo Life Sciences) was used to detect APs[5 (link)]. Briefly, CAMs (1×105/ml) were collected and centrifuged (400 × g, 5 minutes) at the end of the treatment. Then, CAMs were incubated with 0.5 mL of freshly diluted Cyto-ID Green Detection Reagent (1:4000) for 30 minute at 37°C in the dark. Without washing, stained CAMs were run in the green (FL1) channel with a Guava Easycyte Mini Flow Cytometry System (Guava Technologies, Hayward, CA) and analyzed with Guava acquisition and analysis software (Guava Technologies). The enhancement of Cyto-ID Green dye signal indicates an increase in APs.
In addition, acridine orange (Sigma) was used to detect APLs. CAMs (1×106/ml) were stained with acridine orange (1:5000) for 17 minutes. After washes, CAMs were harvested in phenol red-free growth medium. Green (510–530 nm) and red (>650 nm) fluorescence emission from 104 cells illuminated with blue (488 nm) excitation light was measured with Flow Cytometry System and analyzed with Guava acquisition and analysis software. The mean red/green fluorescence ratio was calculated to indicate the change of intracellular APLs.
Xu M., Li X.X., Chen Y., Pitzer A.L., Zhang Y, & Li P.L. (2014). Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes. Journal of Cellular and Molecular Medicine, 18(11), 2165-2175.
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