Facs gallios flow cytometer

Manufactured by Beckman Coulter

Sourced in United States

The FACS Gallios flow cytometer is a high-performance instrument designed for multi-parameter analysis of cells and particles. It utilizes laser-based technology to detect and measure the physical and fluorescent characteristics of individual cells or particles as they pass through a flow cell. The FACS Gallios is capable of analyzing a wide range of sample types, including cells, microbeads, and other biological particles.

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7 protocols using facs gallios flow cytometer

Flow Cytometric Analyses of Cell Death and Cell Cycle

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For all flow cytometric analyses, a minimum of 10,000 cells were analyzed for each sample. We used a FACS Gallios flow cytometer (Beckman Coulter), and data were analyzed with the FlowJo software package. The FACS gating and analysis strategies are shown in Supplementary Fig. 13. Additional details are given in the following paragraphs.
Cell death assays: Following a specific treatment, mouse and human cells were harvested by trypsinization, washed in phosphate-buffered saline (PBS), and resuspended in 100–200 μl PBS containing propidium iodide (PI; 2.5 μg/ml) for 15–20 min at room temperature (RT) before flow cytometric analysis.
Cell cycle analyses: Cells were harvested as detailed above. Next, cells were fixed with 70% ice-cold ethanol, washed in PBS, treated with 100 μg/ml RNase A at RT for 5 min, then incubated with 50 μg/ml PI for 15–20 min at RT before flow cytometric analysis. Apoptotic cells were identified by the quantitation of the SubG0 (<2n DNA) cell population.

Bhattacharya K., Maiti S., Zahoran S., Weidenauer L., Hany D., Wider D., Bernasconi L., Quadroni M., Collart M, & Picard D. (2022). Translational reprogramming in response to accumulating stressors ensures critical threshold levels of Hsp90 for mammalian life. Nature Communications, 13, 6271.

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Apoptosis Assessment by Flow Cytometry

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Cell lines were seeded into 6-well plates (1 × 10⁵ cells/well). Twenty-four-hour later Staurosporine (Sigma Aldrich, USA, S4400) or DMSO control was added to the medium. Twenty-four-hour later supernatant was collected, cells were trypsinized (Fisher Scientific, USA, Cat. No. 11560626), washed with ice-cold PBS, and resuspended in antibody-binding buffer (10 mM HEPES pH 7.4 (Fisher Scientific, USA, Cat. No. 15630080), 140 mM NaCl; 2.5 mM CaCl₂). Cells were stained for Annexin-V (BD Biosciences, USA, Cat. No. 556420) and 50 µg/mL propidium iodide (Carl Roth, Germany, Cat. No. CN74). After 20 min of incubation in the dark, samples were analyzed on a FACS Gallios Flow Cytometer (Beckman Coulter). We used FACS Kaluza software (Beckman Coulter) to quantify populations. At least 5 × 10⁴ events were assessed per measurement. All measurements were performed as duplicates. Gates used can be found in Supplementary Fig. 8. Data are presented as mean ± SD.

Schaufler D., Ast D.F., Tumbrink H.L., Abedpour N., Maas L., Schwäbe A.E., Spille I., Lennartz S., Fassunke J., Aldea M., Besse B., Planchard D., Nogova L., Michels S., Kobe C., Persigehl T., Westphal T., Koleczko S., Fischer R., Weber J.P., Altmüller J., Thomas R.K., Merkelbach-Bruse S., Gautschi O., Mezquita L., Büttner R., Wolf J., Peifer M., Brägelmann J., Scheffler M, & Sos M.L. (2021). Clonal dynamics of BRAF-driven drug resistance in EGFR-mutant lung cancer. NPJ Precision Oncology, 5, 102.

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Comprehensive Immune Cell Profiling

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After PBMC purification, 1 million cells were pre-blocked by incubation with 10% normal human serum at RT for 15 min and then stained with 50μl of the PANEL Ki-67 antibody cocktail against cell surface markers (anti-CD45RO-PE, -CD19-ECD, -CD56-PC7, -CD3-APC, -CD45RA-APCAlexaFluor750 and -CD16-KO antibodies) (BD Biosciences, Beckman). Cells were washed twice with Staining Buffer and resuspended in 250μl BD Cytofix/Cytoperm solution at 4°C for 20 min. Cells were washed twice in BD Perm/Wash solution. Next, cells were fixed/permeabilized in 50μl BD Perm/Wash solution containing an antibody cocktail against intracellular markers (anti-GzB-AlexaFluor700, -Ki-67-V450) as described in the figures at 4°C for 30 minutes in the dark. Cells were washed twice in BD Perm/Wash solution and resuspended in Staining Buffer prior to flow cytometric analysis on a Beckman Coulter FACS Gallios flow cytometer using the Kaluza software. Events were initially gated on forward and side scatter (SSC) to identify lymphocytes. A bivariate plot of CD56 versus CD3 was used to acquire at least 10,000 NK cells.

Krzywinska E., Cornillon A., Allende-Vega N., Vo D.N., Rene C., Lu Z.Y., Pasero C., Olive D., Fegueux N., Ceballos P., Hicheri Y., Sobecki M., Rossi J.F., Cartron G, & Villalba M. (2016). CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity. PLoS ONE, 11(4), e0150434.

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Apoptosis and Cell Cycle Analysis by Flow Cytometry

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Apoptosis was measured by flow cytometry following Annexin V and propidium iodide staining on a FACS Gallios Flow Cytometer and the corresponding Kaluza analysis software (Beckman Coulter, USA). Cell-cycle analyses were performed by flow cytometry on methanol-fixed cells after propidium iodide staining. For FACS analysis of cleaved caspase-3 (CC3) and γH2AX, cells were treated for indicated times, harvested by trypsinization, and fixed in 80% methanol. Fixed cells were permeabilized and blocked with PBS/1% BSA before they were incubated with primary antibodies at 4°C overnight. The following day, cells were washed, incubated with Alexa Fluor secondary antibodies (Thermo Scientific), and measured on a Gallios Flow Cytometer (Beckman Coulter, USA).
For clonogenic survival assays, cells were seeded in 6-well plates, treated for indicated times, fixed with 4% formaldehyde, and stained with crystal violet solution. For quantification, a 1% SDS solution was added to the wells for 30 min and absorption was measured at 590 nm in the supernatant.

Brägelmann J., Dammert M.A., Dietlein F., Heuckmann J.M., Choidas A., Böhm S., Richters A., Basu D., Tischler V., Lorenz C., Habenberger P., Fang Z., Ortiz-Cuaran S., Leenders F., Eickhoff J., Koch U., Getlik M., Termathe M., Sallouh M., Greff Z., Varga Z., Balke-Want H., French C.A., Peifer M., Reinhardt H.C., Örfi L., Kéri G., Ansén S., Heukamp L.C., Büttner R., Rauh D., Klebl B.M., Thomas R.K, & Sos M.L. (2017). Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma. Cell Reports, 20(12), 2833-2845.

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Apoptosis Induction by MASM

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Cells were seeded and allowed to attach overnight and then incubated with different concentrations of MASM for 24 h. Apoptosis was detected using Annexin V-FITC/PI Apoptosis Detection kit (BD biosciences, San Jose, California, USA) and analyzed by flow cytometry with FACS Gallios flow cytometer (Beckman coulter).

Zou Y., Sarem M., Xiang S., Hu H., Xu W, & Shastri V.P. (2019). Autophagy inhibition enhances Matrine derivative MASM induced apoptosis in cancer cells via a mechanism involving reactive oxygen species-mediated PI3K/Akt/mTOR and Erk/p38 signaling. BMC Cancer, 19, 949.

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NK Cell Cytotoxicity Assay with K562 Targets

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After PBMC purification and NK cell quantification, 3 million cells were incubated at 37°C for 4h or overnight with K562 target cells at an Effector (NK cell): Target ratio of 1:10 in a final volume of 500μl (RPMI Glutamax with 10% FBS and 10u/ml IL2). The medium also contained 1.5μl anti-CD107a antibody (BD Biosciences, Franklin Lakes, NJ) and 1μl monensin to prevent CD107a degradation (BD Golgi-Stop BD Biosciences). Then, cells were resuspended in 50μl of an antibody cocktail containing 7AAD, the anti-CD45RO-FITC, -CD69-PE, -CD19-ECD, -CD56-PECy7, -CD3-APC, -CD45RA-APCAlexaFluor750, -CD107a-HV500 and -CD16-KO antibodies (BD Biosciences, Beckman). Samples were analyzed on a Beckman Coulter FACS Gallios flow cytometer using the Kaluza software. Events were initially gated on forward and side scatter (SSC) to identify lymphocytes. A bivariate plot of CD56 versus CD3 was used to acquire at least 10,000 NK cells.

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Multiparameter Flow Cytometry Analysis

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Thirty-eight fluorescently labelled multimers (streptavidinphycoerythrin (PE), streptavidin-allophycocyanin (APC), and fluorescein isothiocyanate (FITC)) covering HLA-A*02:01, A*24:02, A*30:01, A*30:02, A*68:01, B*58:01, and C*07:01 presenting peptides from the MTB-derived proteins Rv0288, Rv1886c, Rv3875, Rv2958c, Rv2957, and Rv0447c, were either constructed in-house (as previously described 22 ) or commercially acquired (Beckman Coulter, San Diego, USA, and Immudex, Copenhagen, Denmark). In the CD3+CD8+CD4À compartment, multimer-positive events were recorded using anti-CD3-PE/Texas red (ECD) (Clone UCHT1; Beckman Coulter), anti-CD4-Pacific orange (Clone S3.5; Invitrogen, Carlsbad, CA, USA), and anti-CD8a-APC/Cy7 (Clone SK1; Becton Dickinson, Franklin Lakes, NJ, USA). Cells in the CD3+CD8ÀCD4+ compartment were excluded from enumeration of multimer-positive events. All cellular analyses were performed using a FACS Gallios Flow-cytometer (Beckman Coulter). Only multimer responses that were at least three times higher than the negative control and for which we could detect more than 50 events were analyzed further.

Axelsson-Robertson R., Rao M., Loxton A.G., Walzl G., Bates M., Zumla A, & Maeurer M. (2015). Frequency of Mycobacterium tuberculosis-specific CD8+ T-cells in the course of anti-tuberculosis treatment. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 32.

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