In vivo migration assays were performed as previously described [19 (link)]. Briefly, LPS-stimulated BMDC from Actin GFP knock in mice (Inhα +/+) and BMDC from Inhα -/- mice stained with Cell Trace Violet (CTV) (Life Technologies), according to manufacturer’s instructions, were mixed 1:1 in sterile PBS. Viability of labeled cells previous to and after the in vivo transfer was 85–90%. A total of 2 x 106 cells in 50μl were injected in left footpad and, as a control, 50μl of PBS in right footpad of 6–9 week-old female C57BL/6 mice. After 48 hours, popliteal lymph nodes were extracted and stained with viability dye Aqua Zombie (Invitrogen) and anti-CD11c PE (Tonbo).
Celltrace violet ctv
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, France, Australia
CellTrace Violet (CTV) is a cell labeling dye manufactured by Thermo Fisher Scientific. It is a fluorescent dye that can be used to track and monitor cell division in various cell-based applications.
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Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (8 mentions)
Facsaria 2, by BD (6 mentions)
Lsrfortessa, by BD (5 mentions)
B cell isolation kit, by Miltenyi Biotec (4 mentions)
Dynabeads mouse t activator cd3 cd28, by Thermo Fisher Scientific (4 mentions)
Cytoflex, by Beckman Coulter (4 mentions)
Interleukin 2 (il 2), by R&D Systems (3 mentions)
Facsaria 3, by BD (3 mentions)
Ovalbumin (ova), by Merck Group (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Anti cd3, by Thermo Fisher Scientific (3 mentions)
Anti biotin microbead, by Miltenyi Biotec (3 mentions)
Facscanto 2, by BD (3 mentions)
Lsr fortessa cytometer, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Gm csf, by R&D Systems (3 mentions)
Aria 2 cytometer, by BD (3 mentions)
Flica poly caspase assay kit, by ImmunoChemistry Technologies (3 mentions)
215 protocols using celltrace violet ctv
1
In Vivo Migration Assay of BMDCs
2
Tracking PBMC proliferation in co-culture
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Peripheral blood mononuclear cells (PBMCs) were labeled with CellTrace Violet (CTV, 5 μM) (Life Technologies) to track proliferation and activated with 2.5 μg/mL phytohemagglutinin (PHA) (Sigma). hISCs or BM-MSCs were seeded in 96 well-plates at four densities (5 × 103, 104, 2 × 104, or 4 × 104 cells/well) in respective media during 24 h to allow cells to adhere. Then, activated PBMCs were added at the concentration of 2.105 cells/well on hISCs or BM-MSCs in co-culture medium consisting in IMDM, 25 mM HEPES, supplemented with 10% inactivated fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM non-essential amino-acids, 0.25 mM beta-mercaptoethanol, 2 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. As control condition, activated PBMCs were cultured alone. After 96 h of co-culture, PBMCs were harvested and proliferation was quantified by flow cytometry using a FACSCanto cytometer (BD Bioscience) on the basis of CTV dilution. Results are expressed as the percentage of proliferation of stimulated PBMCs and normalized to 100% for activated PBMCs alone.
3
In Vivo Cytotoxicity Assay for Influenza
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In vivo cellular cytotoxicity assay were adapted from Durward et al. (30 (link)). In brief, splenocytes were harvested and single cell suspensions prepared. Splenocytes were incubated with the MHC class I restricted influenza HA (Cal07) peptide IYSTVASSL, the NP peptide (RLIQNSLTIERMVLS), or a negative control peptide, at a density of 5x107 cells/mL for 1 h at 4°C followed by 30 min incubation at 37°C. Peptide‐loaded cells were washed twice in PBS and subsequently stained with 5 μM CellTrace Violet (CTV) (C34557, Life Technologies) (HA peptide loaded cells), or 1 μM CellTrace Far Red (CTFR) (C34564, Life Technologies) (NP peptide loaded), or double stain (CTV and CTFR) (negative control) at a density of 5x107 cells/mL for 20 min at 37°C. Cells were mixed in equal ratios (1:1:1), and a total of 15x106 cells injected i.v. in a 100µl volume to vaccinated mice. Spleens were harvested 16h later, single cell suspensions prepared, and the presence of peptide loaded cells investigated by flow cytometry. The ratio of CTV to CTV/CTFR or CTFR to CTV/CTFR cells were calculated as % specific lysis = [1 − (average ratio in group with NaCl vaccinated mice/experimental ratio)].
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In vivo cellular cytotoxicity assay were adapted from Durward et al. (30 (link)). In brief, splenocytes were harvested and single cell suspensions prepared. Splenocytes were incubated with the MHC class I restricted influenza HA (Cal07) peptide IYSTVASSL, the NP peptide (RLIQNSLTIERMVLS), or a negative control peptide, at a density of 5x107 cells/mL for 1 h at 4°C followed by 30 min incubation at 37°C. Peptide‐loaded cells were washed twice in PBS and subsequently stained with 5 μM CellTrace Violet (CTV) (C34557, Life Technologies) (HA peptide loaded cells), or 1 μM CellTrace Far Red (CTFR) (C34564, Life Technologies) (NP peptide loaded), or double stain (CTV and CTFR) (negative control) at a density of 5x107 cells/mL for 20 min at 37°C. Cells were mixed in equal ratios (1:1:1), and a total of 15x106 cells injected i.v. in a 100µl volume to vaccinated mice. Spleens were harvested 16h later, single cell suspensions prepared, and the presence of peptide loaded cells investigated by flow cytometry. The ratio of CTV to CTV/CTFR or CTFR to CTV/CTFR cells were calculated as % specific lysis = [1 − (average ratio in group with NaCl vaccinated mice/experimental ratio)].
4
Adoptive Transfer of Tumor-Specific T Cells
5
T cell proliferation with MSCs
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Fresh splenocytes or purified CD4+ T cells were labeled with CellTrace Violet (CTV) (Life-Technology, Saint Aubin, France) prior to being cocultured with or without WT MSC or Gilz-/- MSC at a 1:10 MSC:T cell ratio in presence of 5 µg/mL of concanavalin (ConA) (Sigma-Aldrich). After 72 h, the proliferation of T cells was quantified by flow cytometry.
6
Isolation and Expansion of Memory-like NK Cells
7
Treg-Mediated Suppression of Colitis
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Tregs from control and Esrrg-cKO splenocytes were isolated, either purified using Treg isolation kit II (Miltenyi Biotec) with the AutoMACS Pro (Miltenyi Biotec) or sorted as FOXP3-YFP+ with the S3e Cell Sorter (Bio-Rad). CD45.1+CD4+CD25– Teff cells from B6.SJL mice were labeled with CellTrace Violet (CTV) (Life Technologies, Thermo Fisher Scientific). Dendritic cells (DCs) were isolated from B6N mice by positive selection with anti-CD11c isolation kit (Miltenyi Biotec). CD45.2+CD4+CD25+ Tregs were incubated with Teff cells (6.6 × 104) at a 1:1 to 1:4 ratio in the presence of DCs (1 × 104) and soluble anti-CD3e antibody (1 μg/mL, BD Biosciences 145-2C11) for 3 days (74 (link)). The proliferation of CD45.1+ Teff cells was determined by the dilution of CTV, and the proliferation index was calculated with the FlowJo software.
For the experimental colitis model, B6.Rag–/– mice were injected i.v. with sorted CD4+FOXP3– Teff cells (0.4 × 106) either alone or with control or Esrrg-cKO Tregs (0.1 × 106). Mice were weighed and examined weekly and euthanized at week 13. Splenocytes were analyzed with flow cytometry, and colons were fixed and stained with H&E or anti-CD45 (1:25 dilution; BD Pharmingen 30F11). Infiltrating lymphocytes in colon were quantified with Imagescope.
For the experimental colitis model, B6.Rag–/– mice were injected i.v. with sorted CD4+FOXP3– Teff cells (0.4 × 106) either alone or with control or Esrrg-cKO Tregs (0.1 × 106). Mice were weighed and examined weekly and euthanized at week 13. Splenocytes were analyzed with flow cytometry, and colons were fixed and stained with H&E or anti-CD45 (1:25 dilution; BD Pharmingen 30F11). Infiltrating lymphocytes in colon were quantified with Imagescope.
8
Splenocyte Proliferation Assay
9
Treg cell-mediated suppression of effector T cells
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Current and exTreg cells were sorted based on expression of YFP and RFP. Naive effector cells (CD4+CD25−CD44loCD62Lhi) cells were sorted from spleens and peripheral LNs of CD45.1 mice and were labeled with cell trace violet (CTV) (Life Technologies) according to manufacturer’s instruction. Cell sorting was done using FACSAria (BD Biosciences). Feeder cells were prepared from spleens of B6 mice and CD4 T cells were depleted using CD4+ magnetic beads (Miltenyi Biotec, Auburn, CA) and then were irradiated with 3000 rads. Feeders were cultured with naive effector T cells at a ratio of 3:1 in U-shaped 96 well plates. Sorted current or exTreg cells were added at a ratio of 1:1, 1:2, 1:4, 1:8 or not added for effectors only wells. All wells, except for unstimulated wells, were stimulated with soluble αCD3 antibody at 2.5 μg ml−1. All cells were cultured in RPMI media supplemented with 10% FCS, L-glutamine, penicillin and streptomycin, β-mercaptoethanol. Cells were harvested 4 days following stimulation and the percentage of effector CD45.1 cells that diluted CTV was determined.
10
Evaluating Treg-mediated Suppression of CD8+ T Cells
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Naive CD45.2+CD8+ T responder cells (Tresp) were negatively sorted using the EasySep Mouse CD8+ T Cell Isolation kit (STEMCELL Technologies Inc.) and stained with CellTrace Violet (CTV; Life Technologies, Carlsbad, CA). CD45.1+CD4+FoxP3-EGFP− T cells that had experienced HD Lm ΔactA-Ova infection for 24 h in C57BL/6 mice were re-isolated using the EasySep Mouse CD4+ T Cell Isolation kit (STEMCELL Technologies Inc.) followed by fluorescent cell sorting CD45.1+CD45.2−FoxP3-EGFP+ and CD45.1+CD45.2−FoxP3-EGFP− cells. 2.5 × 104 CD45.2+CD8+ Tresp were placed in co-culture with graded numbers of sex-matched, magnetic/fluorescent cell sorted CD45.1+FoxP3-EGFP+ (Treg) or EGFP− (conventional) CD4+ T cells and 1.25 mL washed Mouse T-Activator anti-CD3/CD28 Dynabeads (GIBCO) in a 96-well round-bottom plate for 72 h. Cell proliferation of CD45.2+CD8+ Tresp was determined by flow cytometry based on dilution of CTV. Percent suppression was calculated as the percent difference in absolute number of dividing Tresp in wells containing EGFP+ or EGFP– CD4+ T cells to the average absolute number of dividing Tresp in wells without addition of EGFP+ or EGFP– CD4+ T cells. Replication index, representative of only Tresp that had responded to anti-CD3/CD28 stimulation, was also determined for the cultures.
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