Trypan blue dye (Thermo Fisher Scientific) exclusion technique for counting of living cells manually was performed using bifocal light microscope before and after T-cell stimulation assays. Cell proliferation was monitored by carboxyfluorescein succinimidyl ester (CFSE) labeling (Thermo Fisher Scientific) at final concentration 1 μM on day 0 and CFSE-labeled cells were stimulated 7 days according to aAPC approach. Dead cells were excluded by 7-amino-actinomycin (7AAD) (BioLegend) staining in combination with the following cell surface antibodies: anti-CD8-PE/Cy7 (SK1) (BioLegend), anti-CD3-APC (SK7) (BD). CFSE dilution and 7AAD were analyzed by flow cytometry.
Cfse labeling
Manufactured by Thermo Fisher Scientific
CFSE labeling is a fluorescent labeling technique used to track cell division and proliferation. It involves the covalent staining of cells with the dye carboxyfluorescein succinimidyl ester (CFSE), which binds to cellular proteins and is distributed equally between daughter cells during cell division. This allows for the quantification of cell division history through flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trypan blue dye, by Thermo Fisher Scientific (1 mentions)
7 amino actinomycin 7 aad, by BioLegend (1 mentions)
Anti cd3 apc sk7, by BD (1 mentions)
Paramagnetic beads, by Miltenyi Biotec (1 mentions)
Cellquest, by BD (1 mentions)
Modfit software, by BD (1 mentions)
Apc conjugated anti cd3 antibody, by BioLegend (1 mentions)
5 protocols using cfse labeling
1
T-cell Proliferation Monitoring by CFSE
2
Coculture protocol for cell transfer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For coculture experiments, tumor sender cells were plated at 1*103 cell in a 96 round bottom plate. Twenty-four hours later, following tumor cell proliferation, receivers were plated with the following conditions: NIH/3T3 receivers at 2*103/well, BMDM receivers at 5*103 cells/well, and CD8+ T cells 15*103/ well. Following 1 min spin at 250 g, cells were incubated in triplicate for indicated time points and harvested and analyzed for BFP expression by flow cytometry. Experiments using CFSE labeling (ThermoFisher) were performed according to the manufacturer’s instructions.
3
Ova-specific Transgenic T Cell Trafficking
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ova-specific transgenic T cells were isolated from CD45.1+ OT-I or CD45.1+ OT-II mice by positive selection of CD8+ or CD4+ T cells using paramagnetic beads (Miltenyi Biotec). Purity (≥95%) was assessed regularly by flow cytometry. After CFSE labeling (Thermo Fisher Scientific), 106 CD8+ OT-I or 2 × 106 CD4+ OT-II T cells were i.v. injected into congenic CD45.2+ recipient mice. 16 h later, targeting antibodies were i.p. injected in different amounts as indicated in the experiments. Mice were sacrificed, and splenocytes were analyzed for proliferation of the transferred transgenic T cells. In case of depletion of specific cell populations, mice were i.v. injected with PBS or 20 ng/g body weight of DT (Merck or Sigma-Aldrich) 24 h before antibody injection. For depletion of pDCs, 200 µg αPDCA-1 antibody was i.p. injected 72, 48, and 24 h before injection of the targeting antibodies.
4
CFSE-based Cell Proliferation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were starved overnight, and cell proliferation (cell division) was assayed using carboxyfluorescein succinimidyl ester (CFSE) labeling (Invitrogen). Briefly, the cells were trypsinized, washed twice in sterile PBS and resuspended in 2 mL of DMEM. CFSE was applied at a final concentration of 5 μM, and the cells were incubated at 37°C for 10 min in the dark. To quench the labeling, the cells were washed twice with complete DMEM. The labeled cells were treated with vehicle or the indicated concentrations of L-4F (5, 10, or 20 μg/mL) for 48 h in complete DMEM. Next, the cells were trypsinized and washed in PBS. Finally, the cells were assessed for labeling using a FACSCalibur flow cytometer. The acquired data were analyzed using CellQuest and Modfit software (BD Biosciences).
5
T Cell Proliferation Assay with Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were activated with 5 µg mL−1 prebound anti‐CD3/anti‐CD28 antibodies (BioLegend), followed by CFSE labeling (Invitrogen) according to the manufacturer's instructions. The activated PBMCs were cocultured with organoids and treated with a conditioned medium from organoids or lung cancer cell lines. After 96 h, the cells were stained with an APC‐conjugated anti‐CD3 antibody (BioLegend), and T cell proliferation was determined by assessing CFSE intensity via flow cytometry.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!