CT26 tumor-bearing mice were treated with 50,000 ppm gNO, 20,000 ppm gNO or nitrogen. Fourteen days post gas treatment all tumors were resected. Mouse spleens were dissociated at day 21 post gas treatment with the GentleMACS Octo (Miltenyi-Biotech). Mouse blood samples were processed 21 days post gas treatment using RBC Lysis Solution (cat#130-094-183, Miltenyi-Biotech). Extracellular labeling of T-cells was performed with FITC anti-CD3 (cat#130-119-758, Miltenyi-Biotech), VioGreen anti-CD4 (cat#130-118-693, Miltenyi-Biotech), and APC-Vio770 anti-CD8 (cat#130–120-806, Miltenyi-Biotech) antibodies. Extracellular labeling of B-cells was performed with PE-Vio615 anti-CD19 (cat#130-111-890, Miltenyi-Biotech) antibodies. Extracellular labeling of polymorphonuclear myeloid-derived suppressor cells (MDSCs) was performed with FITC anti-CD3 (cat#130-119-758, Miltenyi-Biotech) and PE-Vio615 anti-CD19 (cat#130-111-890, Miltenyi-Biotech) for negative staining, VioGreen anti-CD11 (cat#130-113-811, Miltenyi-Biotech), PE Ly-6G (cat#130-123-780, Miltenyi-Biotech) antibodies for positive staining and VioBlue anti-Ly6C (cat#130-111-921, Miltenyi-Biotech) for low staining. Samples were analyzed using MacsQuant™ 16 Flow cytometer.
Anti cd3 fitc
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD3-FITC is a fluorescently-labeled antibody that binds to the CD3 receptor on T cells. It is commonly used for flow cytometric analysis of T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8 apc, by Miltenyi Biotec (3 mentions)
Anti cd4 viogreen, by Miltenyi Biotec (3 mentions)
Anti cd4 pe, by Miltenyi Biotec (2 mentions)
Anti cd19 fitc, by Miltenyi Biotec (2 mentions)
Facscalibur, by BD (2 mentions)
Slyntvatl, by Immudex (2 mentions)
Anti cd8 apc sk1, by BD (2 mentions)
Ymldlqpett, by Immudex (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Anti cd8 vioblue, by Miltenyi Biotec (2 mentions)
Facsverse, by BD (2 mentions)
Anti cd4 apc vio770, by Miltenyi Biotec (2 mentions)
Anti cd8 apc vio770, by Miltenyi Biotec (2 mentions)
Gentlemacs octo, by Miltenyi Biotec (2 mentions)
Anti cd16 cd32 antibody, by BioLegend (1 mentions)
Anti cd4 fitc, by Miltenyi Biotec (1 mentions)
Anti cd25 apc, by Miltenyi Biotec (1 mentions)
Anti cd44 apc, by Miltenyi Biotec (1 mentions)
Facssuite, by BD (1 mentions)
Anti cd8 pe, by Miltenyi Biotec (1 mentions)
17 protocols using anti cd3 fitc
1
Evaluating Immune Responses in Tumor-Bearing Mice
2
Flow Cytometry Analysis of Immune Cells
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Unpurified splenocytes or isolated subpopulations were subjected to flow cytometric analyses to validate the quality of the cells. Prior to staining, the Fc receptors were blocked by preincubation with anti‐CD16/CD32 antibodies (Biolegend, San Diego, CA, USA) for 10 minutes on ice. Surface staining was accomplished by incubating the cells with fluorochrome‐conjugated specific antibodies for 20 minutes in the dark on ice. The following antibodies (all purchased from Miltenyi Biotec) were employed: anti‐CD3‐FITC (130‐102‐496), anti‐CD4‐FITC (130‐102‐541), anti‐CD4‐PE (130‐102‐619), anti‐CD8‐PE (130‐102‐595), anti‐CD19‐FITC (130‐092‐042), anti‐CD25‐APC (130‐102‐787), anti‐CD44‐APC (130‐102‐563), anti‐CD62L‐PE (130‐102‐907).
Intracellular staining of FoxP3 was performed using an anti‐FoxP3‐PE antibody (130‐098‐119; Miltenyi Biotec) and the FoxP3 Staining Buffer Set (130‐093‐142; Miltenyi Biotec) following the given instructions.
Flow cytometric analyses were run on a FACS Verse (BD Biosciences) or FACS Calibur (BD Biosciences). A total of 10 000 events per sample were acquired and data were evaluated using the FACS Suite or CellQuest Pro software (both BD Biosciences).
Intracellular staining of FoxP3 was performed using an anti‐FoxP3‐PE antibody (130‐098‐119; Miltenyi Biotec) and the FoxP3 Staining Buffer Set (130‐093‐142; Miltenyi Biotec) following the given instructions.
Flow cytometric analyses were run on a FACS Verse (BD Biosciences) or FACS Calibur (BD Biosciences). A total of 10 000 events per sample were acquired and data were evaluated using the FACS Suite or CellQuest Pro software (both BD Biosciences).
3
Peripheral Lymphocyte Immunotyping Protocol
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We quantified the CD 19 + lymphocytes (B cells), T CD3 + lymphocytes, T CD3 + CD4 + lymphocytes, T CD3 + CD8 + lymphocytes and CD56 + CD3- cells (NK cells). A polychromatic flow cytometry tube was used for peripheral lymphocyte immunotyping developed at the immunology laboratory of the National Medical Genetics Center (Zúñiga Rosales et al., 2020 ). The monoclonal antibodies conjugated with fluorochromes from MACS MiltenyiBiotec (Germany) included anti-CD45 APC-Vio770 (Clone 5B1), anti-CD19 PE-Vio700 (Clone LT19), anti-CD3 FITC (Clone BW264/56), anti-CD4 PerCP-Vio700 (Clone M−T466), anti-CD8 APC (Clone BW135/80), anti-CD56 PE (Clone REA196), (Fig. S1 ).
4
Isolation of Immune Cell Subsets from CSF
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The index patients’ parents have given written informed consent, and analyses were approved by the Charité University Hospital Institutional Review Board.
4 ml of CSF was collected during the acute phase of encephalitis (Nikolaus et al., 2018 (link)) and immediately processed for cell pellet cryopreservation, therefore centrifuged for 10 min at 400× g, with supernatant stored at −80°C and the pellet resuspended in 500 µl of 10% dimethyl sulfoxide, 45% fetal calf serum, 45% RPMI medium before freezing at −80°C. PPE was collected 6 d after lumbar puncture and stored at −80°C.
We used fluorescence-activated cell sorting to isolate single CD138+ ASCs, CD20+CD27+ MBCs, and CD20+CD27− NMBCs from preselected viable CD3−CD14−CD16−DAPI− lymphocytes into 96-well PCR plates. The following antibodies were applied: anti–CD3-FITC (1:25; Miltenyi Biotec; #130-098-162), anti–CD14-FITC (1:25; Miltenyi Biotec; #130-098-063), anti–CD16-FITC (1:25; Miltenyi Biotec; #130-098-099), anti–CD20-PerCP-Vio700 (1:50; Miltenyi Biotec; #130-100-435), anti–CD27-APC-Vio770 (1:12.5; Miltenyi Biotec; #130-098-605), and anti–CD138-PE (1:50; Miltenyi; #130-098-122).
4 ml of CSF was collected during the acute phase of encephalitis (Nikolaus et al., 2018 (link)) and immediately processed for cell pellet cryopreservation, therefore centrifuged for 10 min at 400× g, with supernatant stored at −80°C and the pellet resuspended in 500 µl of 10% dimethyl sulfoxide, 45% fetal calf serum, 45% RPMI medium before freezing at −80°C. PPE was collected 6 d after lumbar puncture and stored at −80°C.
We used fluorescence-activated cell sorting to isolate single CD138+ ASCs, CD20+CD27+ MBCs, and CD20+CD27− NMBCs from preselected viable CD3−CD14−CD16−DAPI− lymphocytes into 96-well PCR plates. The following antibodies were applied: anti–CD3-FITC (1:25; Miltenyi Biotec; #130-098-162), anti–CD14-FITC (1:25; Miltenyi Biotec; #130-098-063), anti–CD16-FITC (1:25; Miltenyi Biotec; #130-098-099), anti–CD20-PerCP-Vio700 (1:50; Miltenyi Biotec; #130-100-435), anti–CD27-APC-Vio770 (1:12.5; Miltenyi Biotec; #130-098-605), and anti–CD138-PE (1:50; Miltenyi; #130-098-122).
5
Multi-parameter Immunophenotyping of MAIT Cells
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For immunophenotyping, cells were stained in PBS supplemented with 0.5% BSA and 2mM EDTA for 15min at 4°C with following fluorochrome-conjugated antibodies: anti-CD69-FITC or anti-CD3-FITC, anti-TCR Vα7.2-PE, anti-CD161-APC (all from Miltenyi Biotec, Germany), anti-CD223(LAG3)-FITC, anti-CD154(CD40L)-FITC (Biolegend, USA). Dead cells were excluded using propidium iodide (Miltenyi Biotec, Germany). MAIT cells were identified based on their TCR Vα7.2 and CD161 expression. Cell surface expression was analyzed on a FACS Calibur (BD Bioscience). Cell sorting was carried out using FACS ARIA II (BD Bioscience). Data were analyzed using FlowJo™ Software Version 10.5.3 (Becton, Dickinson and Company).
6
Isolation of DCs and T cells from Splenic Lymphocytes
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Splenic lymphocytes were collected and cultured as described previously55 (link). Part of splenic lymphocytes was incubated with anti-CD11c microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and separated using AutoMacs (Miltenyi Biotec). Selected cells were considered CD11c+ DCs. Negatively selected cells were stained with anti-CD3 FITC (Miltenyi Biotec), and subsequently incubated with anti-FITC microbeads (Miltenyi Biotec) to obtain CD3+ T cells. DCs and CD3+ T cells were cultured in RPMI-1640 medium supplemented with 10% FBS for further detection of the percentages of PRRSV-specific CD3+ T cells producing IFN-γ and IL-4.
7
Flow cytometric analysis of immune cell populations
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Example 3
The following reagents were used: anti-CD3 FITC (Clone BW264/56, Miltenyi Biotec), anti-CD8-APC (SK1, BD Pharmingen), Dextramer-HPV E711-20-PE (Dext-HPV PE)(HLA-A*0201; YMLDLQPETT, Immudex), Dextramer-HIV-1 P17 Gag 77-85-PE (Dext-HIV-PE)(HLA-A*0201; SLYNTVATL, Immudex), anti-TCR-Vß3-FITC (JOVI-3, Ancell). Cells were incubated with Dext as recommended by the manufacturer for 10 min at room temperature, then mAb were added for 30 min at 4° C. Stained cells were washed with PBS/0.5% BSA/0.1% NaN3 (PBA), and were analyzed using a FACSCalibur flow cytometer (Becton Dickinson).
8
Intracellular GZMA Expression in Sepsis
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After 24 h of sepsis induction, WT and GZMA-/- mice were sacrificed. Blood and spleen were collected aseptically. PBLs were isolated from blood as described above and spleen was homogenized in 5 mL of RPMI medium. 2x105 PBLs or splenocytes were stained with anti CD3-FITC, anti CD8-APC, anti NK1.1-APC-Vio770 and anti CD4-VioBlue from Miltenyi Biotec. Subsequently, cells were fixed with paraformaldehyde (PFA) 1%, permeabilised with saponin 1% in PBS and incubated with anti gzmA-PE (eBioscience) or with the corresponding isotype control (IgG2b kappa isotype control PE, eBiosciencie). Finally, intracellular expression of GZMA was analysed by FACS.
9
Comprehensive Immune Cell Phenotyping
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Stimulations were stopped by incubation in 2mM EDTA for 5 min. Surface staining was performed for 15 min in the presence of 1 mg/ml of Beriglobin (CSL Behring) with the following fluorochrome-conjugated antibodies titrated to their optimal concentrations as specified in Supplementary Table 2
10
Immune Cell Biomarker Analysis by Flow Cytometry
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We assessed the levels of biomarkers on the surfaces of the immune cells from the blood and ascites samples by flow cytometry.19 (link) Briefly, each specimen underwent erythrocyte lysis with red blood cell lysis buffer, then washing with PBS. Then, we incubated the specimens (100 μL) with either anti-CD3-FITC, anti-CD19-percp-cy5.5, anti-Siglec-10-APC (Catalog number: 130-103-731, clone: 5G6, Miltenyi Biotec, Bergisch Gladbach, Germany), and anti-CD150-PE (eBioscience, San Diego, CA, USA) or anti-PD-1-PE (eBioscience) and anti-TIM-3-APC (eBioscience) at working concentration. Then, the specimens were incubated in the dark at 4 °C for 20 min, re-suspended, and washed twice in PBS. Flow cytometry was performed using a FACSCanto™ II flow cytometer (BD Biosciences, San Diego, CA, USA). The data were analyzed with FlowJo software (Treestar, San Carlos, CA, USA). Antigen expression is depicted as fluorescence intensity on dual-parameter scattergrams.
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