Supernatants (cRF5) were collected from CHO cells expressing devil 41BB extracellular domain fused to either mCherry (pAF137), mCitrine (pAF138), mOrange (pAF164), mBFP (pAF139), mAzurite (pAF160), mCerulean3 (pAF161), or mNeptune2 (pAF163) (tables S2 to S4). The supernatant was spun for 10 min at 3200 RCF to remove cells and cellular debris and then stored at 4°C until further use. CHO cells expressing devil 41BBL (CHO.pAF56) and untransfected CHO cells were prepared as described above. Flow cytometry tubes were loaded with 5 × 104 target CHO cells per well in cRF5, centrifuged 500 RCF for 3 min, and then resuspended in 200 μl of supernatant from the 41BB FAST cell lines (N = 1 per treatment). The tubes were then incubated for 15 min at 4°C, centrifuged at 500 RCF for 3 min, resuspended in 400 μl of cold fluorescence-activated cell sorting (FACS) buffer, and stored on ice until the data were acquired on a Beckman Coulter Astrios flow cytometer (Fig. 2C ). All flow cytometry data were analyzed using FCS Express 6 Flow Cytometry Software version 6 (De Novo Software).
Astrios flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Astrios flow cytometer is an analytical instrument designed to detect and analyze particles, cells, and other components in a fluid sample. It utilizes the principles of flow cytometry to provide measurements on the physical and chemical characteristics of individual particles or cells within the sample. The core function of the Astrios is to enable researchers and scientists to gather quantitative data on the properties of their samples, such as size, granularity, and fluorescence, which can be used for a variety of applications in fields such as immunology, cell biology, and diagnostics.
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Lab products found in correlation
Anti cd31 pe, by BD (1 mentions)
Apc anti cd49f, by BD (1 mentions)
Anti cd44 apc, by BD (1 mentions)
Anti cd45 pe, by BD (1 mentions)
Anti cd24 fitc, by BD (1 mentions)
Fitc conjugated donkey anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
8 well chamber slide, by Ibidi (1 mentions)
5 protocols using astrios flow cytometer
1
Characterization of 41BB Fusion Proteins
2
Analyzing CHO Cell Responses
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U-bottom 96-well plates were loaded with 1 × 105 target CHO cells per well in cRF5, centrifuged 500 RCF for 3 min, and then resuspended in 175 μl of cRF5 supernatant from FAST cell lines collected 11 days after transfection (N = 1 per treatment). The plates were then incubated for 30 min at room temperature, centrifuged at 500 RCF for 3 min, resuspended in 200 μl of cold FACS buffer, centrifuged again, and fixed with FACS fix buffer [PBS, 0.02% NaN3, 0.4% formalin, glucose (10 g/liter)]. The cells were transferred to tubes, diluted with FACS buffer, and analyzed on a Beckman Coulter Astrios flow cytometer (fig. S2).
3
Flow Cytometry Analysis of Stem Cell Markers
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Flow cytometry was performed using an Astrios flow cytometer (Beckman coulter, Inc. Brea, CA, USA). Conjugated mouse anti-human antibodies APC anti-ESA, APC anti-CD49f, APC anti-CD44, FITC anti-CD24, PE anti-CD45, PE anti-CD31, PE anti-CD235 and anti-mouse H2KD were purchased from BD Pharmingen (San Diego, CA, USA). Unconjugated mouse anti-human antibody CD10 (BD Pharmingen) was used with FITC conjugated donkey anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were harvested by dissociation using 0.05% trypsin/EDTA. 1 × 106 cells were resuspended in 200 μl HBSS with 2% FBS and then stained with the proper amount of antibodies (according to the instruction sheet) for 30 minutes at 4°C. Cells incubated with unconjugated antibodies were stained with secondary antibodies for another 30 min at 4°C. For the Aldefluor assay, the ALDEFLUOR kit (StemCell Technologies Inc., Vancouver, BC, Canada) was used and perform as previously described [21 (link)]. Dead cells were excluded by DAPI staining (0.2 μg/ml). Stem cell percentages in tumor cells were analyzed after gating out H2KD positive mouse cells.
4
Isolation and Characterization of Plasma Microvesicles
5
Cellular Uptake of Fluorescent Polyplexes
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Polyplexes were prepared using FITC-labelled polymers and Cy 3 -labelled plasmid DNA to study the cellular uptake and internalisation. For confocal microscopy, A549 and H1299 cells were seeded into 8-well chamber slides (obtain from Ibidi) at a seeding density of 1 Â 10 4 cell per cm 2 in 300 ml of fully supplemented medium. After 24 hours, the cells were washed with PBS, covered with 300 ml of FBS-free media, and treated with labelled polyplexes with an N/P ratio of 10 at a concentration of 2 mg ml À1 of DNA for 4 hours. Then, the cells were washed with PBS three times, fixed with 4% paraformaldehyde for 15 minutes, stained with DRAQ5 (20 mM in PBS for 5 minutes), and covered with mounting medium. In the case of flow cytometry, the cells were prepared under conditions similar to the transfection experiment. After incubation of the cells with labelled polyplexes for 4 hours, the cells were washed with PBS three times, and detached from the plates using trypsin solution for 5 minutes at 37 1C. Then, the cells were collected, centrifuged, and re-suspended in 0.3 ml of PBS in FACS tubes. Thereafter, the samples were mixed with 150 ml of 0.4% trypan blue and measured using an Astrios flow cytometer from Beckman Coulter Life Sciences.
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