CD34+ CB cells (1 × 106 cells/ml) were labeled with 5 μl carboxyfluorescein diacetate succinimidyl ester (Biolegend, Cat: 423801) according to the manufacture’s guidelines. Labeled cells were cultured for 24 h before they were stained with APC-labeled anti-human CD34 antibody (Biolegend, Cat: 343608) and sorted for CD34+CFSE+ cells using a FACS MoFlo (Beckman). Sorted cells were then resuspended in HSC expansion medium supplemented with DMSO (0.01%) or JNK-IN-8 (2 µM). Cell aliquots after 2 and 4 days in culture were stained with APC anti-human CD34 (Biolegend, Cat: 343608) and APC-Cy7 anti-human CD45RA antibody (Biolegend, Cat: 304128) before cells were analyzed for CFSE intensity by FCS express version 6 software.
Facs moflo
Manufactured by Beckman Coulter
Sourced in United States
The FACS MoFlo is a high-performance flow cytometry instrument designed for cell sorting and analysis. It offers advanced features for precise and efficient cell separation and data collection. The core function of the FACS MoFlo is to enable the sorting and analysis of complex cell populations based on their physical and fluorescent characteristics.
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Lab products found in correlation
Facscalibur, by BD (2 mentions)
Carboxyfluorescein diacetate succinimidyl ester, by BioLegend (1 mentions)
Anti human cd34 apc, by BioLegend (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Edu flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Summit software, by Beckman Coulter (1 mentions)
Annexin 5 apc 7 aad apoptosis detection kit, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Anti human foxp3 staining set, by Thermo Fisher Scientific (1 mentions)
Helios clone 22f6, by BioLegend (1 mentions)
Ifnγ clone b27, by BioLegend (1 mentions)
Cd4 clone 13b8.2, by Beckman Coulter (1 mentions)
Il 2 clone mq1 17h12, by BioLegend (1 mentions)
Cd161 clone hp 3g10, by BioLegend (1 mentions)
Foxp3 clone 236a e7, by Thermo Fisher Scientific (1 mentions)
11 protocols using facs moflo
1
CD34+ CB Cell Expansion and Characterization
2
Cell Cycle Analysis of NPSCs
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NPSCs were trypsinised, collected and washed twice with PBS. Then, single cell resuspensions were fixed in pre-cooled 70% ethanol and stored at − 20 °C for later use. For cell cycle analysis, the fixed cells were centrifuged, washed twice with PBS containing 2% (v/v) FBS and resuspended in PBS. Subsequently, the resuspended cells were incubated in 500 μl staining solution containing Hoechst 33342 (for DNA content, 2 μg/mL) and Pyronin Y (for RNA content, 1 μg/mL) for 20 min at room temperature. Then, cells were placed on ice before flow cytometry analysis, and 10,000 events were collected per sample with FACS MoFlo (Beckman, USA).
3
Isolation and RNA extraction of CD45+CD90+ cells
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Cells were incubated for 20 min at room temperature (RT) with allophycocyanin (APC) conjugated anti-CD45 IgG1 (J33) (Beckman Coulter, Brea, CA, USA), and fluorescein isothiocyanate (FITC) conjugated anti-CD90 IgG1 (5E10) (BD Biosciences, Franklin Lakes, NJ, USA) or appropriate isotype controls. Cells were washed with PBS and sorted by FACS MoFlo (Beckman Coulter) in culture medium supplemented with 10% heat inactivated FBS. After one wash with PBS, cells were resuspended in 1 mL Trizol® (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) and stored at −80 °C.
For the most accurate sorting we chose the “purified mode” and a droplet envelope of one drop. The sorting speed was around 5000 cells/s.
For the most accurate sorting we chose the “purified mode” and a droplet envelope of one drop. The sorting speed was around 5000 cells/s.
4
NPC Cell Cycle Analysis by EdU Flow Cytometry
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To determine the cell cycle of NPC, total cell‐cycle progression was assessed using EdU Flow Cytometry Assay Kits (Invitrogen, Massachusetts, USA). Single NPC suspensions were prepared in PBS, and the cells were treated with the corresponding specialized reagent. The cell pellets were incubated at 37°C for 30 min and obtained by centrifugation. Finally, the specimens were investigated by flow cytometry with FACS MoFlo (Beckman, USA).
5
Cell Surface and Apoptosis Marker Analysis
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For the analysis of cell surface markers, cells were trypsinised and incubated for 20 min with murine anti-human antibodies, the information of antibodies was listed in Table S5 . The expression of intracellular markers (Bcl2 and active caspase-3) was detected by incubating the permeabilised cells with Alexa Fluor 647-conjugated murine anti-human Bcl2 or active caspase-3 (Table S5 ).
For apoptosis analysis, an annexin V-APC/7-AAD apoptosis detection kit (BD Pharmingen, USA) was used following the manufacturer’s instructions. We took the total proportion of annexin V-positive cells (quadrants II and III) as the apoptotic rate, regardless of the 7-AAD status. FACS analysis was performed on a FACS Calibur (BD Biosciences, USA), and data analysis was performed using FlowJo Software (TreeStar, USA).
CD133+ and SP cells were sorted for the analysis of CSLC. The staining procedure of SP cells was described previously [26 (link)]. Hoechst 33342-effluxing cells were analysed and sorted by ultraviolet laser cytometry (FACS MoFlo, Beckman Coulter, USA), while CD133+ cells were sorted on a FACS Aria III (BD Biosciences, USA). Data analysis was performed using Summit Software (Beckman Coulter, USA).
For apoptosis analysis, an annexin V-APC/7-AAD apoptosis detection kit (BD Pharmingen, USA) was used following the manufacturer’s instructions. We took the total proportion of annexin V-positive cells (quadrants II and III) as the apoptotic rate, regardless of the 7-AAD status. FACS analysis was performed on a FACS Calibur (BD Biosciences, USA), and data analysis was performed using FlowJo Software (TreeStar, USA).
CD133+ and SP cells were sorted for the analysis of CSLC. The staining procedure of SP cells was described previously [26 (link)]. Hoechst 33342-effluxing cells were analysed and sorted by ultraviolet laser cytometry (FACS MoFlo, Beckman Coulter, USA), while CD133+ cells were sorted on a FACS Aria III (BD Biosciences, USA). Data analysis was performed using Summit Software (Beckman Coulter, USA).
6
Multicolor Flow Cytometry of T Cell Subsets
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For multicolor flow cytometric analysis, PBMCs were stained with the following fluorescence-conjugated monoclonal antibodies against CD4 (clone 13B8.2; Beckman Coulter Inc., Indianapolis, IN, USA), CD25 (clone M-A251; Becton Dickinson), CD49d (clone 9F10; Becton Dickinson), CD127 (clone HIL-7R-M21; Becton Dickinson), CD152 (clone L3D10; Biolegend, San Diego, CA, USA), and CD161 (clone HP-3G10; Biolegend). Cells were then fixed and permeabilized using the anti-human Foxp3 staining set (eBioscience, San Diego, SC, USA) followed by intracellular staining with monoclonal antibodies against Foxp3 (clone 236A/E7; eBioscience), IFN-γ (clone B27; Biolegend), IL-2 (clone MQ1–17H12; Biolegend), IL-17 (clone N49–653; Biolegend), and Helios (clone 22F6; Biolegend) according to the manufacturer’s instructions. The cells were analyzed on a FACS Calibur (Becton Dickinson) or FACS MoFlo (Beckman Coulter Inc.). In some experiments, the expression level of Foxp3 was evaluated using the mean fluorescent intensity (MFI).
7
Isolation and Characterization of AT2 and BASCs
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AT2 and BASCs sorting was performed as previously described [12 (link)]. (The number of wild-type mice used for cell sorting n = 6). Briefly, single lung cells suspension was stained with the following anti-mouse antibodies (all antibodies were provided by BD, Franklin Lakes, NJ, USA): APC anti-CD45 (Clone: 30-F11), APC anti-CD31 (Clone: MEC 13.3), and PE-Cy7 anti-CD326 (EP-CAM) (Clone: G8.8) and PE anti-Ly-6A/E (Clone: E13-161.7). After staining, cells were washed once and re-suspended in PBS containing 2% FBS. AT2 (CD31 negative, CD45 negative, Sca-1 negative, Ep-CAM positive) and BASCs (CD31 negative, CD45 negative, Sca-1 positive, Ep-CAM positive) were sorted with the use of FACS MoFlo (Beckman Coulter, Brea, CA, USA), (Figure 1 ). In addition, after staining according to the same protocol as for cell isolation using FACS, BrdU staining and analysis using flow cytometry were performed.
8
Retroviral Expression of HOIP Mutants
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Coding sequences of HOIP-WT, HOIP-C885S, HOIP-N102A, deletion mutants of human HOIP fused or not at the C terminus to the TAP-tag and TAP-tagged NOD2 (aa 28–1040) were inserted into the retroviral MSCV vector containing GFP as selection marker. Upon infection, cells were sorted using MoFlo FACS (Beckman Coulter).
9
Retroviral Transduction and FACS Sorting
10
Retroviral Transduction and FACS Sorting
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Coding sequences of HOIP-WT, HOIP C885S, TBK1 WT or TBK1 D135N were inserted into the retroviral MSCV vector containing GFP as selection marker. Upon infection, cells were sorted using MoFlo FACS (Beckman Coulter).
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