Dissociated cells were seeded on round-bottom 96-well plates and washed with 200 μl of ThermoFisher’s PBS buffer 1x (428 x g, 4 °C for 5 min). Twenty μl of Biolegend Zombie Aqua fixable viability stain (1:500) was added and incubated for 20 min in the dark at 4 °C. Excess viability stain was washed away with 200 μl of PBS/BSA/EDTA buffer (428 x g, 4 °C for 5 min) and the pellet resuspended and incubated for 10 min at 4 °C in 4 μl of Biolegend’s FcR blocking antibody (1:10). Cells were then incubated for 30 min at 4 °C with surface staining antibody master mix. After washing free unbound antibody with 200 μl of PBS (428 x g, 4 °C for 5 min), cells were fixed with 100 μl of Biolegend’s fixation buffer 1x for 20 min at 4 °C in the dark. Following fixation, cells were permeabilized and washed with 100 μl of Biolegend perm-wash solution 1 x (428 x g, 4 °C for 5 min) and the pellet resuspended and incubated for 30 min at 4 °C in intracellular staining antibody master mix (antibody dilution 1/100). Stained cells were placed in 200 μl of PBS/BSA/EDTA 1x and visualized on a BD Biosciences FACS Aria II using FACS Diva software. Analysis was performed using BD Biosciences FlowJo software. Antibody panels are described in supplemental material (Supplementary Table 2 ).
Perm wash solution
Manufactured by BioLegend
Sourced in United States
Perm-wash solution is a buffer designed for use in flow cytometry applications. It is used to permeabilize cells and wash away unbound antibodies during intracellular staining procedures.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2, by BD (2 mentions)
Fcr blocking antibody, by BioLegend (2 mentions)
Zombie aqua fixable viability stain, by BioLegend (2 mentions)
Facscalibur, by BD (1 mentions)
Cytoflex lx flow cytometer, by Beckman Coulter (1 mentions)
Anti cd4 fitc, by BioLegend (1 mentions)
Anti cd45 bv421, by BioLegend (1 mentions)
Zombie nir dye, by BioLegend (1 mentions)
Anti ki67 fitc, by BioLegend (1 mentions)
Anti cd4 bv711, by BioLegend (1 mentions)
Anti cd11b bv510, by BioLegend (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Cytexpert 2, by Beckman Coulter (1 mentions)
Fixation permeabilization solution, by Thermo Fisher Scientific (1 mentions)
Perm wash solution, by Thermo Fisher Scientific (1 mentions)
Pe cy7 anti cd8a, by BioLegend (1 mentions)
4 protocols using perm wash solution
1
Multiparametric flow cytometry
2
Exosome Effects on HL-60 Cell Proliferation
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The Ki-67 protein is a nuclear antigen related to cell proliferation that can be used as a marker for cell proliferation assays. To determine the effect of BM-MSC exosomes on HL-60 cells’ proliferation, first, HL-60 cells (seeded in 12-well plates at a density of 12 × 104 cells/well), treated with 0 and 100 μg/mL of exosomes (3 samples × each dose) for 24 h at 37 °C, were collected, washed with 1 mL of PBS 1X and centrifuged for 5 min at 1500 rpm. After that, 100 μL of fixation reagent was added to the tube and incubated at RT for 30 min. The cells were washed twice with 1 mL of Perm/Wash solution (BioLegend, USA). Then, 100 μL of Perm/Wash solution along with Ki-67 antibody was added to the samples, incubated for 30 min at 4 °C, and washed twice with 1 ml of Perm/Wash solution again. The final step was analyzing samples with BD FACS Calibur (BD Biosciences, San Jose, CA, USA).
3
Multicolor Flow Cytometry for Cell Viability
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Cell pellets obtained from the striatum and midbrain were resuspended in 100 µL of CB and incubated for 5 min at RT with Zombie NIR Dye (BioLegend, San Diego, CA, USA) to assess cell viability (50 µL/sample; 1:2000 dilution in PBS). After quenching with CB, cells were centrifuged at 2000 rpm for 1 min, and labeled with BV510 anti-CD11b (1:500, M1/70, BioLegend), BV421 anti-CD45 (1:1000, 30F11, BioLegend), PE-Cy7 anti-CD8a (1:400, 53 − 6.7, BioLegend) or BUV395 anti-CD8a (1:200, 53 − 6.7, BD Bioscience), and FITC anti-CD4 (1:1000, GK1.5, BioLegend) or BV711 anti-CD4 (1:200, GK1.5, BioLegend) diluted in CB and the FcR Blocking Reagent (1:50; Miltenyi Biotec, Bergisch Gladbach, Germany) during 15 min at 4 °C in the dark. For intracellular staining, cells were incubated with fixation/permeabilization solution (Invitrogen) during 7 min at 4 °C in the dark and washed with PermWASH solution (Invitrogen) and incubated with FITC anti-Ki67 (1:500, 11F6, BioLegend) diluted in PermWASH solution during 15 min at 4 °C in the dark. Once labeled, samples were centrifuged, resuspended in CB and data was acquired using a CytoFLEX LX Flow Cytometer (Beckman Coulter, Brea, CA). The analysis was performed using the CytExpert 2.3 (Beckman Coulter) and the FlowJo 10.0.7r2 (BD Biosciences, Franklin Lakes, NJ) softwares.
4
Dengue Virus Envelope Protein Detection
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Cells were transferred to V-bottom 96-well plates and washed with 200 μl of PBS buffer 1× (428 × g, 4°C for 5 min). 20 μl of Zombie Aqua fixable viability stain (Biolegend, 1 : 500) was added and incubated for 20 min in the dark at 4°C. Excess viability stain was washed away with 200 μl of PBS/BSA/EDTA buffer (428 × g, 4°C for 5 min) and the cell pellet resuspended and incubated for 10 min at 4°C in 4 μl of FcR blocking antibody (Biolegend) (1/10). Cells were then incubated for 30 min at 4°C with surface staining antibody master mix (Supplementary Table 1 ). After washing free unbound antibody with 200 μl of PBS (428 × g, 4°C for 5 min), cells were fixed with 100 μl fixation buffer (Biolegend) 1× for 20 min at 4°C in the dark. Following fixation, cells were permeabilized and washed with 100 μl of perm-wash solution 1× (Biolegend, 428 × g, 4°C for 5 min) and the cell pellet resuspended and incubated for 30 min at 4°C with anti-DENV envelop protein antibody (clone 4G2 ATCC HB-112, labeled with Alexa Fluor 488) (Molecular probes; Thermo Fisher) (antibody dilution 1/100). Stained cells were resuspended in 200 μl of PBS/BSA/EDTA 1× and visualized on a BD Biosciences FACS Aria II using FACS Diva software. Analysis was performed using BD Biosciences FlowJo x10.0.7r2 software. Gating strategy is described in Supplementary Fig. 2 .
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