Splenocytes (1x10 6 per ml) from mice treated or untreated with MSCs and infected with Plasmodium were suspended in FACS buffer (PBS with 2% FBS). Surface staining was carried out by incubating with the fluorochrome-labelled antibodies anti-CD4-FITC (fluorescein isothiocyanate) and anti-CD25-APC (allophycocyanin) at 4°C for 30 min in the dark. Cells were fixed for 30 min at RT, in the dark with 2% paraformaldehyde and then treated with Perm/Wash solution (e-bioscience) before incubation with PE (phycoerythrin)-conjugated anti-FOXP-3 antibody (e-bioscience) in Perm/Wash solution at 4°C for 1 hr. Finally, cells were washed in Perm/Wash solution and then washed in FACS buffer and resuspended in FACS buffer. Cells were acquired using LSR Fortessa (BD, Biosciences, USA).
Perm wash solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Perm/Wash solution is a laboratory reagent used in various applications. It serves as a buffer to facilitate the permeabilization and washing of samples during sample preparation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, 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)
Pe cy7 anti cd8a, by BioLegend (1 mentions)
Perm wash solution, by BioLegend (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P erk, by BD (1 mentions)
P shp1, by Cell Signaling Technology (1 mentions)
Pplcγ2, by BD (1 mentions)
P cd79a, by Cell Signaling Technology (1 mentions)
P pi3k p85, by Cell Signaling Technology (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
6 protocols using perm wash solution
1
Characterization of Tregs in Malaria
2
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.
3
Phosphoflow Analysis of B Cell Signaling
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For phosphoflow experiments, 0.5 × 106 cells were cultured in RPMI‐2% FCS at 37°C for 1 min for pPI3K p85 (Cell Signaling Technologies), 3 min for pSHP‐1 (Cell Signaling Technologies), 5 min for pSrc (Abcam), pLyn (Abwiz Bio, San Diego, CA, USA), pCD79a (Cell Signaling Technologies), pSyk, pSLP‐65, pPLCγ2, and pErk (all BD Biosciences) or for 3 h for pS6 and pAkt (both Cell Signaling Technologies) with 25 μg/mL anti‐IgM or antigen‐binding F(ab’)2‐Igκ fragments (anti‐IgK; Jackson Immunoresearch). After fixation with the eBioscience FoxP3 staining kit Fix/Perm solution, cells were washed twice with the accompanying Perm/Wash solution (Invitrogen). Next, cells were incubated with varying combinations of monoclonal antibodies (Supporting information Table S1 ) and stained according to previously described procedures [31 (link)]. After staining for the phosphoproteins, samples were measured in MACS buffer on an LSR‐II (BD Biosciences) and analyzed using FlowJo v10 (BD Biosciences).
4
Multiparameter Flow Cytometry of Immune Cells
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Cryopreserved cells were thawed, washed, and permeabilized with Perm/Wash solution (BD Biosciences). Cells were then incubated at 4°C for 1 h with fluorescence-conjugated antibodies directed against surface antigens and intracellular cytokines. For detection of the transcription factor FOXP3, a nuclear permeabilization buffer (eBioscience, San Diego, CA, USA) was used in place of the Perm/Wash solution as per manufacturer’s protocol. The following fluorescence-conjugated antibodies were used: anti-CD3 PacBlue, anti-γδ TCR PE, anti-CD27 PECy7, anti-CD25 APC, anti-IFNγ Alexafluor 700, anti-IL17A Alexafluor 647, anti-Ki67FITC (BD biosciences, San Jose, CA, USA); anti-CD45RA QDot655, anti-CD8 QDot605, anti-CD4 QDot605 (Invitrogen, Eugene, OR, USA); and anti-CD39 FITC, anti-IL22 PerCP-Efluor710, and anti-FOXP3 PE (eBioscience, SanDiego, CA, USA).
The entire sample was acquired on a BD LSRFortessa Flow Cytometer using FACSDiva software (BD Biosciences, San Jose, CA, USA). Compensation was performed using compensation beads.
The entire sample was acquired on a BD LSRFortessa Flow Cytometer using FACSDiva software (BD Biosciences, San Jose, CA, USA). Compensation was performed using compensation beads.
5
Cell Apoptosis and Cell Cycle Analysis
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Cells were seeded into 6-well plates at a density of 2 × 105 cells/well. Cells either exposed to radiation (8 Gy) or not were treated with DMSO or DQ (1 mM D+20 mM Q) for 24 h, digested with trypsin, and collected. Cells were then resuspended in a 100-μl binding buffer with 1-μl fluorescein isothiocyanate Annexin-V and 1-μl propidium iodine (PI; BD Biosciences, 556547). Finally, samples were analyzed by flow cytometry (C6, BD Biosciences, San Jose, CA). For cell cycle analysis, cells were fixed with Fixation/Permeabilization Diluent/Concentrate (eBioscience) for 30 min. Subsequently, intracellular Ki-67 (eBioscience) and Hoechst33342 (Sigma) staining were performed using PermWash solution (eBioscience). Cells were washed once prior to flow cytometry analysis.
6
TCR Activation and T cell Surface Antigen Analysis
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Splenocytes were isolated and plated in a 96 well plate at 106 cells per well. For TCR stimulation, non-tissue culture treated wells were previously coated with anti-CD3 (145-2C11) antibody at 5μg/ml and then CD28 antibody (37.58) was added to the media at 1μg/ml (BD Biosciences). After 16 hours cells were surface stained with CD8 and CD4 antibodies (4 wells were combined for staining) and then treated with cytofix-cytoperm solution (BD Biosciences) for 15 minutes at 4°C in the dark. Cells were washed two times in Perm Wash solution (eBioscience) and incubated for 15 minutes with mouse Fc-Block (eBioscience) followed by either LFG antibody (Imgenex) or rabbit IgG isotype control (DA1E, Cell signaling) diluted in Perm Wash 5% goat serum at 2.5μg/well. The secondary antibody was goat-anti-rabbit conjugated to Alexa 488 (Molecular Probes).
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