Cells were stimulated by incubation for 5 h with IL-7 (10 ng/ml) alone; or IL-1β (10 ng/ml), IL-23 (10 ng/ml), IL-21 (20 ng/ml) and IL-7 (10 ng/ml); or PMA (50 ng/ml), Ionomycin (500 ng/ml) and IL-7 (10 ng/ml), in the presence of Brefeldin A (5 mg/ml; eBioscience). Cells were washed with 1XHBSS/BSA and incubated with Fc block before staining for surface CD3, TCRγδ, Vγ4, Vγ5, Vγ1, CD27, CD73, and/or CD24 (see flow cytometry section for clones). Cells were fixed and permeabilized (Fix and Perm Cell Permeabilization Kit; eBioscience) and stained with antibodies against IL-17A (clone eBio17B7) or IFNγ (clone XMG1.2). For intranuclear staining for RORγt (clone B2D), cells were fixed and permeabilized (FoxP3 Staining Kit, eBioscience).
Fix and perm cell permeabilization kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fix and Perm Cell Permeabilization Kit is a laboratory reagent designed for the fixation and permeabilization of cells. It facilitates the intracellular staining of proteins, allowing for the analysis of cellular components by flow cytometry or microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (3 mentions)
Alexa fluor 647 donkey anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Fluorofix buffer, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Facs caliber flow cytometer, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Cytofix cytoperm buffer, by Thermo Fisher Scientific (1 mentions)
2.4g2 anti fc receptors, by BD (1 mentions)
Anti il 4 pe, by BD (1 mentions)
Monensin, by BD (1 mentions)
Anti ifn γ apc cy7, by BD (1 mentions)
Acea novocyte flow cytometer, by Agilent Technologies (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Fortessa x20 cytometer, by BD (1 mentions)
Ki67 clone b56, by BD (1 mentions)
Foxp3 staining buffer set, by Miltenyi Biotec (1 mentions)
18 protocols using fix and perm cell permeabilization kit
1
Cytokine Stimulation and T Cell Analysis
2
Immune Cell Phenotyping and Cytokine Analysis
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Aliquots of 5 × 105 APCs were stained with FITC- or PE-conjugated monoclonal antibodies against rat CD200R, CD80, CD86, or OX6. For staining of Foxp3 Tregs, T cells were incubated for 30 minutes at 4°C with anti-CD4 or isotype control Abs, fixed overnight with 1 mL fixation buffer (Fix and Perm cell permeabilization kit; eBioscience, La Jolla, CA, USA), washed, and incubated for 30 minutes at 4°C with anti-rat-Foxp3 antibody. For intracellular cytokine staining, T cells were pretreated for 4 hours with 50 ng/mL PMA, 1 μg/mL ionomycin, and 1 μg/mL brefeldin A (all from Sigma-Aldrich Corp.), then were washed, incubated for 30 minutes at 4°C with anti-CD4 or isotype control Abs, fixed, permeabilized overnight with Cytofix/Cytoperm buffer (eBioscience), and incubated for 30 minutes at 4°C with anti-rat-IFN-γ or IL-17 Abs. Data collection and analysis were performed on a FACScaliber flow cytometer using CellQuest software (BD, San Jose, CA, USA). All antibodies used for flow cytometry analysis were purchased from eBioscience.
3
Multiparametric Flow Cytometry Analysis
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Cells (1 × 106 cells/sample) were washed with fluorescence-activated cell sorting staining buffer (phosphate-buffered saline, 2% fetal bovine serum or 1% bovine serum albumin, 0.1% sodium azide). All samples were incubated with the 2.4G2 anti-Fc receptors (BD Pharmingen), prior to incubation with other Abs diluted in fluorescence-activated cell sorting buffer supplemented with 2% anti-Fc receptor Ab. Cells were subsequently stained with fluorescence-conjugated anti-mouse CD4, CD8, CD44, and CD62L antibodies (all antibodies from eBioscience). For intracellular cytokine staining, 50 ng/ml PMA and 1 mg/ml Ionomycin (all from Sigma-Aldrich) were added followed by 1 mg/ml brefeldin A and 2 mM monensin three hours later. After another two hours, cells were collected, incubated with the 2.4G2 anti-Fc receptors (BD Pharmingen) and stained with fluorescence-conjugated anti-mouse CD4 antibody (eBioscience). After staining, cells were washed and subsequently fixed for 20 min with 1 ml fixation buffer (Fix and Perm cell permeabilization kit, eBioscience). After washing, the fixed cells were stained with fluorescence-conjugated anti-mouse IFN-γ and IL-17 antibody (all antibodies from eBioscience). Data collection and analysis were performed on a FACS Calibur flow cytometer using CellQuest software.
4
T Cell Cytokine Profiling via ICS
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For the ICS, T cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (2 μg/mL; Sigma-Aldrich) for 5 hrs and with Monensin (1 μl/ml; BD Pharmingen) added for the final 4 hrs. Cells were pre-incubated with mouse IgG (20 μg) to block non-specific binding. Each sample was then labeled with anti-CD4-PerCP-Cy5.5 (1:200 μl) and anti-CD8-PE-Cy7 (1:200 μl) for 30 min. Cells were fixed by addition of 2% paraformaldehyde (100 μl) for 10 min followed by staining buffer (5% BSA and 0.1% sodium azide in PBS). ICS was performed with Fix and Perm cell permeabilization kit (Invitrogen) according to the manufacturer’s instructions. Cells were suspended in permeabilization buffer along with mouse IgG (50 μl). For cytokine staining, cells were incubated with either anti-IL-4-PE or anti-IFN-γ-APC-Cy7 (1:100) for 30 min (BD Biosciences, San Diego, CA). The cells were incubated for 30 min and washed with staining solution (5% BSA and 0.1% sodium azide in PBS). Flow cytometry was performed as described with appropriate gating on the CD4+ and CD8+ populations.
5
Cytokine Analysis of Activated T Cells
6
Cell Cycle Analysis of Leptin and SM-Treated Neuro2a Cells
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For cell cycle analysis, Neuro2a cells after 48 h incubation with leptin (400 ng/mL) and SM (0.1 µM) were detached from 24-well plate with TrypLE Express (Gibco, Grand Island, NY, USA) and fixed in cold 70% ethanol. The cells were resuspended in PBS, and DAPI was added at 10 µg/mL.
Cell marker expression was assessed using the FIX and PERM™ Cell Permeabilization Kit (Invitrogen, Frederick, MD, USA). Leptin/SM-treated Neuro2a cells were collected in a 1.5 mL tube using TrypLE and fixed with Reagent A for 15 min at room temperature (RT). Cells were then washed with PBS containing 5% FBS (5% FBS/PBS) and incubated with antibodies against vimentin, fibronectin, and ZO-1 (1:100 in Reagent B) for 1 h at RT. After washing with 5% FBS/PBS, the cells were stained with Alexa Fluor® 488-conjugated secondary antibody (1:2000 in 2% FBS/PBS) for 30 min in the dark at RT, washed again, and resuspended in 2% FBS/PBS.
Flow cytometry analysis was performed using the ACEA NovoCyte flow cytometer (ACEA Biosciences Inc, San Diego, CA, USA). For each sample, 10,000 events were acquired.
Cell marker expression was assessed using the FIX and PERM™ Cell Permeabilization Kit (Invitrogen, Frederick, MD, USA). Leptin/SM-treated Neuro2a cells were collected in a 1.5 mL tube using TrypLE and fixed with Reagent A for 15 min at room temperature (RT). Cells were then washed with PBS containing 5% FBS (5% FBS/PBS) and incubated with antibodies against vimentin, fibronectin, and ZO-1 (1:100 in Reagent B) for 1 h at RT. After washing with 5% FBS/PBS, the cells were stained with Alexa Fluor® 488-conjugated secondary antibody (1:2000 in 2% FBS/PBS) for 30 min in the dark at RT, washed again, and resuspended in 2% FBS/PBS.
Flow cytometry analysis was performed using the ACEA NovoCyte flow cytometer (ACEA Biosciences Inc, San Diego, CA, USA). For each sample, 10,000 events were acquired.
7
Flow Cytometry Analysis of Brn3b+ Retinal Ganglion Cells
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Single RGLCs and W-RBCs were suspended with a fluorescence-activated cell sorting analysis buffer (PBS, 0.5% BSA, 2 mM EDTA-2Na-2H2O). The FIX and PERM™ Cell Permeabilization kit (Invitrogen; Thermo Fisher Scientific, Inc.) and indirect immunolabeling were applied for flow cytometry analysis according to the manufacturer's protocols. Briefly, RGLCs were incubated with POU class 4 homeobox 2 (Brn3b, 1:100) primary antibody for 15 min at RT, and incubated with rabbit anti-fluorescein isothiocyanate-conjugated secondary antibody (1:300) (both from Abcam, Cambridge, UK) for 20 min at room temperature. The percentage of Brn3b+ cells was detected by flow cytometry (fluorescence-activated cell sorter Aria; BD Biosciences, Franklin Lakes, NJ, USA).
8
Multiparameter Flow Cytometry Immunophenotyping
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Flow cytometric analysis was carried out using the following reagents: Live/dead UV Zombie, KLRG1 APC (2F1), CD4 Brilliant Violet 711 (OKT-4), CD28 Brilliant Violet 510 (CD28.2) and CD45RA PECy7 (HI100) all from Biolegend. CD28 Brilliant Violet 786 (U28), CD3 PECF594 (UCHT1), CD8 APC-H7 (SK1), CD57 Alexa Fluor 421 (NK-1), and human cutaneous lymphocyte antigen (CLA) FITC (HECA-452) from BD Biosciences. Cell suspensions were incubated with antibody solutions for 30 min at 4°C for extracellular staining. Intracellular staining for Ki67 (clone B56, BD Bioscience) was performed with Foxp3 Staining Buffer Set (Miltenyi Biotec, Bisley, UK). Cytokine and anti-hTERT (rabbit IgG—Abcam) staining were performed using Fix and Perm Cell Permeabilization Kit (Invitrogen, Paisley, UK) according to the manufacturer's protocol. Samples were processed at Fortessa X-20 cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar). Isotype control staining and fluorescence-minus-one controls were used to set the quadrants.
9
Cardiac Troponin T Quantification
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Dissociated hiPSC-CMs were transferred to cytometry tubes and washed with DPBS. Fixable live/dead staining was used to remove dead cells from the analysis. FluoroFix buffer (BioLegend) was used before using Fix and Perm Cell Permeabilization Kit (Thermo Fisher Scientific). Anti-Troponin T Cardiac Isoform (clone 13–11; Thermo Fisher Scientific) diluted 1:500 was used to label cTnT and followed by secondary staining with Alexa Fluor® 647 donkey anti-mouse IgG (H + L) (Life Technologies) diluted 1:500. The samples were kept overnight at 4 °C before analysis (BD Fortessa) (N = 3, n = 3).
10
Measuring Transcription Factor Expression in B Cells
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