J-Lat 8.4 cells were latently infected with an HIV-1 defective pseudovirus encoding GFP (26 (link)). Cells were either untreated or treated with TPA for 8hrs, chilled on ice, washed with cold PBS, and kept on ice before sorting. FACS Aria II cell sorter (BD Biosciences) was used to sort GFP+ and GFP− J-Lat 8.4 cells. Singlets were selected by forward versus side scatter profiles.
Facs aria 2 cell
Manufactured by BD
Sourced in United States
The FACS Aria II cell sorter is a high-performance flow cytometry instrument designed for cell sorting applications. It utilizes advanced technology to enable precise and efficient cell analysis and separation. The core function of the FACS Aria II is to provide users with the capability to accurately identify, isolate, and collect specific cell populations from complex samples.
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Lab products found in correlation
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Facscanto, by BD (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
6 protocols using facs aria 2 cell
1
Sorting of HIV-1 Latently Infected Cells
2
Cell Isolation and Sorting Protocol
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The cells were treated with 0.25% trypsin/EDTA for 5 min at 37°C, and dissociated by pipetting in HBSS (GIBCO). Dead cells stained with propidium iodide were excluded from the analysis. The cells were analyzed and sorted using a FACS Aria II cell sorter (BD). The isolated cells were collected in PBS with 2% FBS containing 10 µM Y27632.
3
Isolation and Flow Cytometry of Bone and Renal Cells
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Flow cytometry and cell sorting were performed on a FACS Aria II cell sorter (BD Biosciences) and analyzed using FlowJo software. Long bones or renal grafts were dissected and freed from the surrounding soft tissue, which was then followed by dissociation with mechanical and enzymatic steps. Specifically, the tissue was placed in collagenase digestion buffer supplemented with DNase and incubated at 37°C for 60 min under constant agitation. After collagenase digestion and neutralization, undigested materials were gently triturated by repeated pipetting. Total dissociated cells were filtered through a 70-m nylon mesh and pelleted at 200 ×g at 4°C for 5 min. Cells were resuspended in ACK (ammonium-chloride-potassium) lysing buffer to eliminate red blood cells and centrifuged again at 200 ×g at 4°C for 5 min. The pellet was re-suspended in 100 µl staining media (2% FBS/phosphate-buffered saline [PBS]) and stained with antibodies for at least 30 min at 4°C. The applied FACS antibodies can be found in the 'Key Resources' table. Living cells were gated for a lack of PI (propidium iodide; 1:1000 diluted stock solution: 1 μg/ml in water) fluorescence. Compensation, fluorescence-minus-one control-based gating, and FACS isolation were conducted prior to analysis or sorting using the antibody combinations as indicated in the respective figures and legends.
4
Single-cell sorting of live non-immune cells
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The single-cell suspensions consisting of immune cells and non-immune cells were stained with BB515 mouse anti-human CD45 (564585, BD, CA, USA) at a concentration of 1:200 on ice in the dark for 30 min, washed with WB and centrifuged at 300 g, 4 °C for 5 min. The washing step was repeated twice. Then the cells were resuspended with 500 µl WB and stained with 7-AAD (559925, BD, CA, USA) 1:20 and analyzed on a FACSAria II cell sorter (FACSAria II, BD biosciences, CA, USA). Live non-immune cells (7-AAD negative/CD45 positive cells) were sorted for scRNA-seq.
5
Isolation of Immature Myeloid Cells
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Fresh PBMCs were isolated from healthy individuals. To isolate immature myeloid cells (ImMCs), PBMCs were stained with antihuman CD33, HLA-DR following the method described above. ImMCs were sorted into CD33dimHLA-DR− cells using BD FACS Aria II cell sorting system (BD, Franklin Lakes, New Jersey). Sorted ImMCs were stored in RPMI (Life Technologies, San Diego, CA) and immediately used in the co-incubation study by the following method.
6
Cell Cycle Phase Sorting by Flow Cytometry
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For cell cycle sorting, live cells were stained with Vybrant Violet DyeCycle Stain according to the manufacturers protocol (Thermo Fisher). Cells in different cell cycles phases (G1, S and G2) were sorted in a BD FACS Aria II cell sorter (BD Biosciences, San Jose, CA) using the violet laser (405 nm) for excitation, the pacific blue channel (BP 450/40 nm) for emission and a 100 µm nozzle at a rate of 1500 events/second. Regular cell cycle analysis was performed by standard procedure with RNase A and propidium iodide (Thermo Fisher) and analyzed in a BD FACS Canto (BD Biosciences).
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