Single cell suspensions were generated from tissue by passing through a 70μm cell strainer (lungs were first digested and minced in 0.6 mg/mL type-2 collagenase (Worthington Biochemical) for 1 hour at 37°C to promote tissue breakdown). Cell suspensions were incubated with anti-CD16/32 antibodies (1:200 dilution; eBioscience clone 93) to block Fc interactions. Subsequent extracellular antibody labeling was performed on ice in PBS supplemented with 0.5% BSA (Roche). Viability staining was performed with 7-AAD (1:1000 dilution; BD Bioscience) for live-cell analysis, or an amine reactive fixable viability dye (1:1000 dilution; FVD eFluor780 eBioscience) when cells were to be fixed and permeabilized. For intracellular labeling, cells were fixed and permeabilized with either the FoxP3 fixation buffer kit or IC fixation buffer kit from eBioscience, according to manufacturer recommendations. Intracellular antibody labeling was performed for 30 minutes on ice in permeabilization buffer (eBioscience). All flow cytometry was performed on BD FACS Canto I and Canto II. Flow cytometric data was analyzed in Flow Jo software version 887 (FlowJo LLC).
Ic fixation buffer kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IC fixation buffer kit is a laboratory reagent used for the preparation of cell samples prior to analysis. The kit provides the necessary components to fix and permeabilize cells, which is a crucial step in intracellular (IC) staining and flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (3 mentions)
Golgiplug, by BD (3 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Golgistop, by BD (3 mentions)
Zombie aqua fixable viability kit, by BioLegend (2 mentions)
Anti fcγrii fcγriii 2.4g2, by BD (2 mentions)
Tnfα mp6 xt22, by Thermo Fisher Scientific (2 mentions)
Fvd efluor780, by Thermo Fisher Scientific (1 mentions)
Canto 2, by BD (1 mentions)
Facscanto 1, by BD (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd16 32 antibody, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Roche (1 mentions)
7 aad, by BD (1 mentions)
Tnf α fitc, by BioLegend (1 mentions)
Il 2 pe, by BioLegend (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Fortessa flow cytometer, by Thermo Fisher Scientific (1 mentions)
6 protocols using ic fixation buffer kit
1
Tissue Dissociation and Single-Cell Flow Cytometry
2
Flow Cytometric Analysis of Immune Cell Subsets
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Cells were washed once in FACS buffer (PBS containing 0.1% BSA) before blocking with anti-FcγRII/FcγRIII (2.4G2, BD Pharmingen). Staining of surface molecules (all Biolegend) was performed on ice using FITC-conjugated CD4 (RM4-5), CD8α (53-6.7), and CD90.1 (OX-7); PECy7-conjugated CD44 (IM7); PE-conjugated α4β7 (LPAM-1, DATK32), CD4 (RM4-5), CD8α (53-6.7), CD62L (MEL-14), CD44 and CD25 (PC61); APC conjugated CD90.1 (OX-7). Intracellular Foxp3 (FJK-16s, APC-conjugated; eBioscience), NFATc1 (anti-NFATc1 PE 7A6, Biolegend), IRF4 (3E4, APC or PB-conjugated; Biolegend) staining was performed using the Foxp3 staining kit (eBioscience) according to the manufacturer’s instructions. Antibodies (all Biolegend) for intracellular cytokine staining were APC-IFN-γ (XMG1.2), FITC-TNF-α (MP6-XT22) and PE-IL-2 (JES6-5H4). Cytokine detection was performed after a 6 h in vitro restimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10 ng/mL, Sigma) plus ionomycin (5 nM, Merck Biosciences) in the presence of GolgiStop and GolgiPlug (both BD Pharmingen) using the IC Fixation Buffer kit (eBioscience). Viable cells were detected with the Zombie Aqua™ Fixable Viability Kit (Biolegend). Data were acquired on a FACSCanto II (BD Biosciences) flow cytometer and analyzed with FlowJo software (Tree Star).
3
Flow Cytometric Analysis of Cytokine Expression
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Cells were washed and blocked in staining buffer (PBS, 0.3% BSA and 0.1% sodium azide) containing the anti-CD16/CD32 antibody for 10 min at 4°C and then stained with fluorophore-conjugated antibodies. After the cells were washed twice with staining buffer, data were collected on a Fortessa flow cytometer (BD Biosciences). For intracellular staining, 2.5 μg/ml BFA was added during the last 4 h of stimulation to block the secretion of cytokines. After the cells were washed and stained for cell-surface markers and fixated and permeabilized with the IC fixation buffer Kit (eBioscience) according to the manufacturer’s protocol, the cells were stained with FITC-anti-mouse IFNγ or isotype control and analyzed with a Fortessa flow cytometer. The data were analyzed using FlowJo software.
4
Flow Cytometric Analysis of Immune Cells
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A total of 1–2 × 106 cells were stained with Zombie Violet viability dye (Biolegend, San Diego, CA, USA) according to the manufacturer specifications and then washed with 500 μL FACS buffer (PBS, 2% FBS, 0.1% sodium azide) before a 10 min incubation with 0.5 µg FcR blocking antibody (2.4G2). The cells were washed with FACS buffer and incubated with a surface antibody cocktail for 15 min on ice. The cells were washed twice before staining intracellularly for MPO using the IC fixation buffer kit from eBioscience (San Diego, CA, USA) in accordance with manufacturer instructions. The cells were re-suspended into FACS buffer and data were acquired from the live cells on a BD LSRII flow cytometer. The data were analyzed with FlowJo software (FlowJo, Ashton, OR, USA).
5
Multiparametric Flow Cytometry Immunophenotyping
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Cells were washed once in flow assisted cell sorting (FACS) buffer [PBS containing 0.1% bovine serum albumin (BSA)] before blocking with anti-FcγRII/FcγRIII (2.4G2, BD Pharmingen). Staining of surface molecules (all Biolegend antibodies) was performed at room temperature using CD4 (RM4-5), CD8α (53-6.7), and CD25 (PC61), TNFRII (TR75-89), and intracellular Foxp3 (FJK-16s, eBioscience). T-bet (4B10, Biolegend) staining was performed using the Foxp3 staining kit (eBioscience) in accordance with the manufacturer’s instructions. The antibodies (all Biolegend) used for intracellular cytokine staining were IFN-γ (XMG1.2); TNF-α (MP6-XT22); IL-2 (JES6–5H4); IL-17A (TC11–18H10.1), and IL-10 (JES5–16E3) using IC Fixation Buffer kit (eBioscience). Cytokine detection was performed after a 6 h in vitro restimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10 ng/mL, Sigma) plus ionomycin (5 nM, Merck Biosciences) in the presence of GolgiStop and GolgiPlug (both BD Pharmingen). Viable cells were detected with the Zombie Aqua™ Fixable Viability Kit (Biolegend). Data were acquired on a FACSCanto II (BD Biosciences) flow cytometer and analyzed with FlowJo software (Tree Star).
6
Comprehensive Immune Cell Profiling
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Total CNS infiltrates, spleen, and LN cells were washed once in FACS buffer (PBS containing 0.1% BSA) before Fc receptor blocking with anti-CD16/CD32 (93; eBioscience, or 2.4G2; BD PharMingen). Staining of surface molecules was performed at room temperature using antibodies against CD4 (RM4-5), CD8α (53-6.7), CD90.1 (OX-7), α4β7 (LPAM-1, DATK32), CD25 (PC61), CD62L (MEL-14), CD69 (H1.2F3), CD45 (30-F11), CD11b (M1/70), PD1 (J43), CD27 (LG.3A10), and TIGIT (Vstm3; differently conjugated; all eBioscience, BioLegend, or BD) in FACS buffer for 15 min in the dark. Intracellular Foxp3 (FJK-16s, PE- or APC-conjugated; eBioscience) staining was performed using the Foxp3 staining kit (eBioscience) according to the manufacturer’s instructions. Intracellular cytokine staining of splenocytes with APC-conjugated IL-2 (JES6-5H4), IFN-γ (XMG1.2), and IL-10 (JES5-16E3) and PE-conjugated IL-4 (11B11), IL-17 (eBio17B7), GM-CSF (MP1-22E9), and TNFα (MP6-XT22; all eBioscience) was performed after 3–6-h restimulation with T/I (tetradecanoyl phorbol acetate [10 ng/ml; Sigma-Aldrich] plus ionomycin [5 nM; Merck Biosciences]) in the presence of GolgiStop and GolgiPlug (both BD Pharmingen) using the IC Fixation Buffer Kit (eBioscience). Data were acquired on a FACS Canto II (BD Biosciences) flow cytometer and analyzed with FlowJo software (TreeStar).
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