Cells were centrifuged at 3000 rpm for 2 min and recovered in blocking buffer (PBS plus 5 per cent FBS and 1 per cent AB human serum (Lonza)). After 20 min of incubation at room temperature, cells were centrifuged and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 min at room temperature. Cells were then washed in Perm/Wash (BD Biosciences) and stained with an anti-alphavirus antibody (clone 1A4B.6) at 1:200 (vol/vol) dilution in Perm/Wash, followed by an Alexa Fluor 488 conjugated anti-mouse IgG antibody (Invitrogen) diluted at 1:400 (vol/vol) in Perm/Wash. Lastly, cells were incubated with a mixture of the following monoclonal antibodies at 1:200 (vol/vol) diluted in blocking buffer: anti-CD4-PE, anti-CD8-PE-Cy5, anti-CD19-PE-Cy7, anti-CD14-BV421 and anti-CD3-APC-Cy7 (BD Biosciences). Cells were washed twice with Perm/Wash between all incubations and were recovered in PBS plus 1.5 per cent paraformaldehyde before flow cytometry analysis on a FACSCanto II cytometer (BD Biosciences).
Perm wash
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Perm/Wash is a laboratory equipment designed for the automated washing and permeabilization of cells. It is used to prepare samples for flow cytometric analysis by gently washing and permeabilizing cells to allow for the intracellular staining of proteins or other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Perm wash, by BD (11 mentions)
Cytofix cytoperm, by BD (7 mentions)
Golgistop, by BD (4 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (3 mentions)
Golgiplug, by BD (3 mentions)
Pe conjugated anti ifn γ, by BD (3 mentions)
Rabbit anti human igg h l f ab 2, by Southern Biotech (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Sodium azide, by Merck Group (2 mentions)
Lsrfortessa, by BD (2 mentions)
Fix perm, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Eq beads, by Standard BioTools (1 mentions)
Helios mass cytometer, by Standard BioTools (1 mentions)
Paraformaldehyde (pfa), by BioLegend (1 mentions)
Aqua live dead dye, by Thermo Fisher Scientific (1 mentions)
Flow staining buffer, by Thermo Fisher Scientific (1 mentions)
Normal mouse serum, by Thermo Fisher Scientific (1 mentions)
Zombie aqua fixable viability dye, by BioLegend (1 mentions)
Anti cd16 32 2.4g2, by BioLegend (1 mentions)
18 protocols using perm wash
1
Intracellular Immune Cell Profiling
2
Cryopreserved Whole Blood CyTOF Protocol
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Whole blood was collected in heparin/lithium tubes and cryopreserved in 10% FCS/DMSO. Samples were rapidly thawed, washed with RPMI medium, and then incubated in RPMI supplemented with 10 mg/mL DNAse for 30 min at 37°C. Cells were then resuspended in 1× PBS and incubated for 15 min at 37°C with 1 μL Intercalator-Rh (DVS Sciences). After washing in PBS-0.5% BSA, cells were stained for 30 min at 4°C, washed with 1× PBS, fixed with PBS-1.6% PFA, and washed with 1× Perm/Wash Buffer (eBiosciences). After permeabilization with 1× Perm/Wash (eBiosciences), intracellular staining was performed for 30 min at 4°C. Cells were washed, fixed in 1.6% PFA, and washed with Barcode Perm Buffer (DVS) before barcoding with a unique combination of three palladium isotopes (DVS, Fluidigm) according to the manufacturer’s recommendations. Cells were then washed with PBS and resuspended in PBS-1.6% PFA containing 0.5 μL Intercalator-Ir (DVS Sciences). Barcoded samples were pooled and stored overnight at 4°C. Before acquisition, cells were washed with milli-Q water and filtered through a cap filter with 35-μm pores (BD Biosciences). Normalization beads (Eq Beads, Fluidigm) were added to the tube and the acquisition was performed using a Helios mass cytometer (Fluidigm). Data were acquired for six consecutive days (3 animals per day).
3
Multiparametric flow cytometry analysis
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Following lysis of red blood cells, cells were washed in PBS and stained with Aqua live/dead dye (Life Technologies, Carlsbad, CA, USA). Prior to surface staining, cells were incubated with 1% mouse serum in flow staining buffer (eBioscience) to block nonspecific binding. Surface markers were stained for CD4, CD8, CD45RO, CD62L and CD3. Following fixation in 4% paraformaldehyde (BioLegend, San Diego, CA, USA), cells were permeabilized in 1× perm/wash (eBioscience) and incubated with antibodies against IFNγ and TNFα. Cells were stored in flow staining buffer at 4 °C prior to acquisition on an LSR II (BD Biosciences, San Jose, CA, USA). Flow cytometry data was analyzed in FlowJo (Tree Star, Ashland, OR, USA) and Excel (Microsoft, Redmond, WA, USA) and graphed in GraphPad Prism (GraphPad Software Inc., LA Jolla, CA, USA).
4
Phosphorylation Profiling of PBMCs
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PBMCs were thawed as described above. After enumeration, PBMCs were incubated for 45min at 37°C+ 5% CO2 to reduce basal phosphorylation levels in warm serum-free RMPI Medium 1640 while in the prescence of the following antibodies (clone): CD3 (OKT3), CD19 (SJ25C1), CD27 (M-T271), CD21 (Bu32), CD11c (B-ly6), and Live/Dead Blue for UV viability dye. Cells were centrifuged 1,500 rpm x 5min at RT, resuspended in warm RPMI with 5% FBS and stimulated with either 10ug/mL Rabbit anti-human IgG (H+L) F(ab’)2 (Southern Biotech, Birmingham, AL) or 75μM pervanadate (used as an experimental positive control) for 5 minutes in a 37°C water bath. After a quick centrifuge spin to pellet the cells (2,400 g x 30 seconds at RT), PBMCs were resuspended in 100uL per million cells Cytofix/Cytoperm (BD) and incubated for 20 minutes on ice. Cells were washed in 1X Perm/Wash (BD), then incubated in the presence of this buffer for intracellular staining for 30min on ice with the following antibodies (clone): pSYK Y348 (I120-722), pPLCγ2 Y759 (K86-689.37) and IgD (IA6-2). Cells were washed with 1X Perm/Wash, centrifuged 1,500 rpm x 5min at 4°C, and resuspended in FACS Buffer containing 1% BSA (Fisher)+ 0.05% Sodium Azide (Aldrich) in 1X PBS. Data was acquired on the Cytek Aurora and cell subsets defined as described above.
5
Signaling Pathways in Activated B Cells
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PBMCs were thawed as described above, then incubated for 45 min at 37°C + 5% CO2 to reduce basal phosphorylation levels while in the presence of the following antibodies (clone) in warm serum-free media: CD3 (OKT3), CD19 (SJ25C1), CD27 (M-T271), CD24 (ML5), CD38 (HB7), CD10 (Hl10a), and viability dye. Cells were then centrifuged at 1,500 rpm for 5 min, resuspended in warm RPMI with 5% FBS, and stimulated with either 10 μg/ml rabbit anti-human IgG (H + L) F(ab’)2 (Southern Biotech) or 75 μM pervanadate (used as an experimental positive control) for 5 min in a 37°C water bath. After a quick spin (2,400 g for 30 s), cells were resuspended in 100 μl per million cells Cytofix/Cytoperm (BD) and incubated for 20 min on ice. Cells were washed in 1× Perm/Wash (BD), then incubated in the presence of this buffer for intracellular staining for 30 min on ice with the following antibodies (clone in parentheses): pSyk Y348 (I120-722), pBlnk Y84 (REA240), pPLCγ2 Y759 (K86–689.37), IgD (IA6-2), and IgM (MHM-88). Cells were washed with 1× Perm/Wash and resuspended in FACS buffer containing 1% BSA (Fisher) + 0.05% sodium azide (Aldrich) in 1× PBS. Data were acquired on the Cytek Aurora and cell subsets defined as described above.
6
Multiparameter Analysis of Immune Cell Subsets
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2 × 106 liver cells, or 20 μl of whole blood was incubated with Zombie Aqua fixable viability dye (Biolegend, London, UK) for 10 min at RT and then with 0.025 μg anti‐CD16/32 (2.4G2; Biolegend) in 10% normal mouse serum (Life Technologies, Paisley, UK). Cells were then incubated with antibodies (Supplemental Table 1). Cells were washed, spun at 300 g for 5 min and, where necessary, incubated with fluorescently labeled streptavidin. 7‐AAD solution (Biolegend) was added to samples 10 min before acquisition when comparing isolation protocols. DAPI was used as a viability marker for FACS. Liver cells were gated as shown, whereas alveolar and interstitial mϕ were identified as CD45+CD11c+SiglecF+ and CD45+CD11c+SiglecF−MHCII+CD64+ cells, respectively.
For BrdU and Ki67 staining, cells were fixed and permeabilized overnight in FoxP3/Transcription Factor Staining Buffer (eBioscience). Cells were washed in PermWash (eBioscience) and stained with anti‐Ki67 and anti‐BrdU antibodies.
Cells were acquired on a LSRFortessa (BD Biosciences, Wokingham, UK) or FACSAriaII (BD) at the QMRI Flow Cytometry and Cell Sorting Facility, University of Edinburgh, and data analyzed in FlowJo software (Tree Star, Ashland, Oregon). Fluorescence‐minus‐one controls were used to set gates.
For BrdU and Ki67 staining, cells were fixed and permeabilized overnight in FoxP3/Transcription Factor Staining Buffer (eBioscience). Cells were washed in PermWash (eBioscience) and stained with anti‐Ki67 and anti‐BrdU antibodies.
Cells were acquired on a LSRFortessa (BD Biosciences, Wokingham, UK) or FACSAriaII (BD) at the QMRI Flow Cytometry and Cell Sorting Facility, University of Edinburgh, and data analyzed in FlowJo software (Tree Star, Ashland, Oregon). Fluorescence‐minus‐one controls were used to set gates.
7
Flow Cytometric Analysis of Regulatory T Cells
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The following anti-human antibodies were used: CD4-PE/Cy5, CD25-FITC, and Foxp3-Alexa647 from BioLegend (San Diego, CA, USA). Mouse cells were stained with fluorochrome-labeled mAbs to CD4-Pacific Blue, Brilliant Violet 605 and Alexa-700 (RM4-5), CD8-Pacific Blue (53-6.7), CD25-APC (7D4), Ki-67-PE (Ki-67), and Thy1.1-PercP (Ox-7) from BioLegend. Foxp3-APC (FJK-16s), dead cell marker Viability Dye eFluor™ 780, and staining reagents from eBioscience (San Diego, CA, USA) were used according to the manufacturer’s instructions. To analyze expression of surface proteins, the cells were stained with the appropriate antibodies for 20 min at 4°C, washed once with FACS buffer (PBS, 0.1% BSA, and 0.02% NaN3), and fixed with 2% paraformaldehyde. For intracellular staining of Foxp3 and Ki-67, the cells were first surface stained, permeabilized with Fix/Perm (eBioscience), and stained with Foxp3 and Ki-67 antibodies diluted in Perm/Wash (eBioscience). To calculate absolute Treg numbers, unlabeled microbeads (BD Biosciences, Franklin Lakes, NJ, USA) were added to the stained cells and the following formula was used: absolute Treg numbers = (beads used × Treg events)/beads measured. Acquisition was performed on a BD™ LSR II or FACSCalibur, and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).
8
Multi-parameter Flow Cytometry Protocol
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Flow cytometry and cell sorting were done on the LSR-Fortessa and FACSAria (BD Biosciences), respectively. Cells were incubated in FACS buffer (5% heat inactivated fetal bovine serum [FBS] [Life Technologies], 2 mM EDTA [Sigma]) in PBS (Life Technologies) with fluorescent-labeled antibodies. Data were analyzed with Flow-Jo 10.2 software (Tree Star) using fluorescent minus one controls for setting gates. Antibodies for mouse studies were obtained from BD Biosciences (Ly6C, CD11b, NK1.1, and CD8), eBioscience (Ki67, F4/80, Foxp3, and CD115), and BioLegend (Ly6G, CD3, CD19, CD4, CD44, CD62L, CD11c, major histocompatibility complex [MHC]-II, IFN-g, tumor necrosis factor [TNF], IL-10, IL-6, immunoglobulin G1 [IgG1], and IgG2a). For intracellular staining, 600,000 cells were incubated in DMEM containing penicillin/streptomycin, 10% FBS, and 2 mM L-glutamine (all Life Technologies) with 10 μg/mL brefeldin A (Sigma), 5 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma), and 500 ng/mL ionomycin (Cayman Chemical) or 1 μg/mL lipopolysaccharide (LPS) (Sigma) for 4 hr. Cells were stained with extra cellular markers and then washed and incubated with Fix/Perm (eBioscience) and washed and incubated with Permwash (eBioscience) and fluorescent-labeled antibodies (IFNγ).
9
Multiparametric Analysis of Mycobacterial Antigen-Specific T Cells
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Individual lung and spleen cells were prepared from immunized and Mtb-infected mice and stimulated with 2.5 µg/mL NG- or G-Ag85A at 37 °C for 12 h in the presence of GolgiPlug and GolgiStop (BD Bioscience, San Jose, CA, USA). First, the cells were washed with 1X PBS (pH 7.4), and the Fc receptor was blocked with an anti-CD16/32-blocking Ab at 4 °C for 15 min. Surface molecules were stained with fluorochrome-conjugated Abs against Thy1.2, CD4, CD8, CD44, and CD62L, and the LIVE/DEADTM Fixable Dead Cell Kit was used for 30 min at 4 °C. The cells were then washed with PBS, fixed, and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 30 min at 4 °C. The permeabilized cells were washed twice with Perm/Wash (BD Biosciences) and stained with PE-conjugated anti-IFN-γ, APC-conjugated anti-TNF-α, and PE-Cy7-conjugated anti-IL-2 Abs for 30 min at 4 °C. The cells were washed twice with Perm/Wash and fixed with IC fixation buffer (eBioscience, San Diego, CA, USA) for flow cytometry analysis.
10
Flow Cytometry Analysis of Immune Cells
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