Transfected HEK 293T cells were collected and disassociated using cold 5 mM EDTA in 1× PBS. Cells were resuspended in 5% fetal bovine serum (FBS), passed through a 26-gauge syringe, and blocked on ice for 30 min. Cells were incubated with either the anti-HA antibody (Sigma), the GP1 antibody directly conjugated to Alexa Fluor 488 (2/11/10-488), or the normal mouse IgG antibody (BioLegend) for 30 min on ice. Cells were pelleted (100 × g, 3 min, 10°C) and extensively washed with 5% FBS. For the HA antibody or normal IgG samples, cells were stained with the Alexa Fluor 488 secondary antibody for 30 min on ice. Cells were washed twice with 5% FBS prior to one wash with 1× PBS. Cells were transferred to prechilled fluorescence-activated cell sorting (FACS) tubes prior to 7-AAD viability staining (BioLegend). Flow cytometry was performed using the FACSCalibur and CellQuest Pro software (BD Biosciences). All data were analyzed using FlowJo software (Ashland, OR).
7 aad viability staining
Manufactured by BioLegend
7-AAD is a fluorescent dye that can be used for cell viability assessment. It is membrane-impermeable and binds to DNA, allowing the identification of dead or dying cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti ha antibody, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
Fixation and permeabilization reagents, by BioLegend (1 mentions)
Fitc anti human cd3 antibody, by BioLegend (1 mentions)
Brefeldin a, by BD (1 mentions)
Fitc conjugated anti cd3, by BioLegend (1 mentions)
Pe conjugated anti cd8a, by BioLegend (1 mentions)
Aurora cs, by Cytek (1 mentions)
Smartstrainer, by Miltenyi Biotec (1 mentions)
Apc cyanine7 conjugated anti cd45, by BioLegend (1 mentions)
Gentlemacs, by Miltenyi Biotec (1 mentions)
Tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Anti cd4 fitc, by BioLegend (1 mentions)
Anti cd45ra fitc, by BioLegend (1 mentions)
Anti cd3 fitc, by BioLegend (1 mentions)
Anti cd4 percp cy5, by BD (1 mentions)
Anti pd 1 fitc, by BioLegend (1 mentions)
Anti ccr7 pe cy7, by BioLegend (1 mentions)
Anti ifn γ apc, by Thermo Fisher Scientific (1 mentions)
5 protocols using 7 aad viability staining
1
Flow Cytometry Analysis of Transfected HEK Cells
2
PD-L1 Expression and Cytokine Secretion in HCC
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The HCC cells were co-cultured with activated T cells for 48h at the ratio of 1:1. Brefeldin A (BD Biosciences) was pretreated with the mixed cells for 6 h. After permeabilizing using fixation and permeabilization reagents (Biolegend 426803), the cells were stained with anti-human CD8-BV510 antibodies (Biolegend 344731), anti-human INF-r-BV421 (Biolegend 506537) and anti-human TNF-a-PE (Biolegend 502908) for 20 mins. Then the samples were ready for flow cytometry.
To analyze membrane PD-L1, HCC cells were dissociated with trypsin-EDTA solution and stained with anti-human CD274 PE antibodies (Biolegend 329705) and incubated for 20 min. Then the cells were harvested for flow analysis.
For PD-L1 flow cytometry from patient samples, we prepared a single-cell suspension from patient samples using MagicFilter and MagicVajra (Bozhentech B160103). Then the suspended cells were stained with APC anti-human CD45 antibodies (Biolegend 304012), Brilliant Violet 510 anti-human CD8 antibodies (Biolegend 344732), FITC anti-human CD3 antibodies (Biolegend 300406), PE anti-human CD274 antibodies (Biolegend 329706), and 7-AAD Viability Staining (Biolegend 420404). After a 20 mins staining, cells were re-suspended in 300 μL of PBS for flow analysis.
To analyze membrane PD-L1, HCC cells were dissociated with trypsin-EDTA solution and stained with anti-human CD274 PE antibodies (Biolegend 329705) and incubated for 20 min. Then the cells were harvested for flow analysis.
For PD-L1 flow cytometry from patient samples, we prepared a single-cell suspension from patient samples using MagicFilter and MagicVajra (Bozhentech B160103). Then the suspended cells were stained with APC anti-human CD45 antibodies (Biolegend 304012), Brilliant Violet 510 anti-human CD8 antibodies (Biolegend 344732), FITC anti-human CD3 antibodies (Biolegend 300406), PE anti-human CD274 antibodies (Biolegend 329706), and 7-AAD Viability Staining (Biolegend 420404). After a 20 mins staining, cells were re-suspended in 300 μL of PBS for flow analysis.
3
Profiling Tumor-Infiltrating T Cells
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To analyze the population
of T cells within the TME, cells were collected from the tumors on
day 10 following various treatments. This was achieved using a tumor
dissociation kit (Miltenyi Biotec, North Rhine-Westphalia, Germany)
along with a dissociator (GentleMACS, Miltenyi Biotec) to obtain single-cell
suspensions, which were then filtered through a 70 μm strainer
(Smartstrainer, Miltenyi Biotec) to eliminate larger debris. Thereafter,
the resulting single-cell suspensions underwent a blocking step with
antimouse CD16/32 antibodies (101302, 1:25, BioLegend) and were subsequently
stained with the following antibodies: APC/cyanine7- conjugated anti-CD45
(103116, 1:40, BioLegend), FITC-conjugated anti-CD3 (100204, 1:25,
BioLegend), PE-conjugated anti-CD8a (100708, 1:100, BioLegend), and
APC-conjugated anti-CD4 (100412, 1:100, BioLegend) antibodies. The
antibody staining process occurred over a period of 30 min at 4 °C,
followed by 7-AAD viability staining (420404, BioLegend). The stained
cells were then analyzed using a cell sorter (Aurora CS, Cytek Biosciences,
Fremont, CA, USA), and the data were analyzed using FlowJo Software
(Treestar, Ashland, OR, USA).
of T cells within the TME, cells were collected from the tumors on
day 10 following various treatments. This was achieved using a tumor
dissociation kit (Miltenyi Biotec, North Rhine-Westphalia, Germany)
along with a dissociator (GentleMACS, Miltenyi Biotec) to obtain single-cell
suspensions, which were then filtered through a 70 μm strainer
(Smartstrainer, Miltenyi Biotec) to eliminate larger debris. Thereafter,
the resulting single-cell suspensions underwent a blocking step with
antimouse CD16/32 antibodies (101302, 1:25, BioLegend) and were subsequently
stained with the following antibodies: APC/cyanine7- conjugated anti-CD45
(103116, 1:40, BioLegend), FITC-conjugated anti-CD3 (100204, 1:25,
BioLegend), PE-conjugated anti-CD8a (100708, 1:100, BioLegend), and
APC-conjugated anti-CD4 (100412, 1:100, BioLegend) antibodies. The
antibody staining process occurred over a period of 30 min at 4 °C,
followed by 7-AAD viability staining (420404, BioLegend). The stained
cells were then analyzed using a cell sorter (Aurora CS, Cytek Biosciences,
Fremont, CA, USA), and the data were analyzed using FlowJo Software
(Treestar, Ashland, OR, USA).
4
Multiparametric Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes
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Specific antibodies against CD3-PE-Cy7 (Biolegend) or anti-CD3-FITC (Biolegend), anti-CD25-PE (BD Pharmingen™), anti-CD127-APC (Biolegend), anti-CD4-FITC (Biolegend), anti-CD8-APC (Biolegend), anti-CD45RA-FITC (Biolegend), anti-CCR7-PE-Cy7 (Biolegend), anti-CD4-PE (BD Biosciences), anti-PD-1-FITC (Biolegend), anti-CD4-PErCP-Cy5.5 (BD Biosciences), anti-Tim-3-PE (BD Pharmingen™), anti-FoxP3-PE (Biolegend), and anti-IFN-γ-APC (eBioscience) were used. 7AAD viability staining (Biolegend) was used to assess the viability of the cells. Tils were examined at day 0, 5, 10, 15, 20 and 25 by flow cytometry. Briefly, 1 × 106 T cells was mixed with 5 μl of each antibody and was incubated on ice for 20 min in the dark. After incubation the samples were washed with FACS buffer (5% BSA in PBS, 0.09% sodium azide). The pellets were suspended in 300 μl of FACS buffer and acquired on a BD FACS Conto II flow cytometer, and then the data was analyzed with FlowJo software (TreeStar Inc). The data is displayed as background-corrected values with control sample or by fluorescence minus one (FMO).
5
Multiparameter Flow Cytometry of Immune Cells
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The following antibodies were purchased from Biolegend: anti-CD3 (clone 17A2), anti-CD45 (clone 30-F11), anti-CD4 (clone GK1.5), anti-CD8α (clone 53-6.7), anti-IFN-γ (clone XMG1.2), anti-CD11b (clone M1/70), and anti-Gr-1 (clone RB6-8C5). The following conjugated antibodies were obtained from eBioscience (San Diego, CA, USA): anti-granzyme B (clone NGZB) and anti-PD-1 (clone J43). Live cells were determined by 7AAD viability staining (Biolegend). For cell surface markers, cells were stained with antibodies at room temperature for 30 minutes. For intracellular antigen detection, an intracellular staining kit containing fixation/permeabilization reagents from eBioscience was used. Flow cytometry analyses were performed on BD LSRFortessa X-20 (BD Biosciences, San Jose, CA, USA).
All gating strategies were determined by fluorescence minus one. Flowjo 10 was used to analyze the flow data.
All gating strategies were determined by fluorescence minus one. Flowjo 10 was used to analyze the flow data.
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