Fluorescent dye

Preparation of CFDA-SE working solution
1.1 Prepare stock solution: it is recommended to dissolve 1 milligram CFDA-SE in 0.1794 mL DMSO to obtain 10 mM CFDA-SE.
Note: Store the stock solution at -20℃ or -80℃ in the dark and avoid repeated freezing.
1.2 Preparation of CFDA-SE working solution.
Dilute stock solution in serum-free cell culture.
Note: Please adjust the concentration of CFDA-SE working solution based on your specific needs.
Cell staining
2.1 Suspended cells: Centrifuge at 1000 g for 3-5 minutes at 4°C and discard the supernatant. Use PBS to wash twice, for five minutes each time.
Adherent cells: Discard the cell culture media, apply trypsin to separate the cells and produce a single cell suspension. Centrifuge at 1000 g for 3-5 minutes at 4°C and discard the supernatant. Use PBS to wash twice, for five minutes each time.
2.2 Add 1 mL CFDA-SE working solution and mix for 30 minutes.
2.3 Centrifuge at 400 g for 3-4 minutes at 4°C.
2.4 Wash the cells twice with PBS, five minutes/each time.
2.5 Resuspend the cells in serum-free medium or in PBS, and detect using fluorescence microscopy or flow cytometer.

CFDA-SE is an intracellular and green fluorescent dye that may be used to detect strain colonization in vivo.
Method: For determine strain colonization in the intestine.
1. Strains are resuspended in PBS to obtain a cell density of 108 CFU/mL.
2. CFDA-SE (1 mM; 10 μL; 20 min; 37°C; dark/protect from light) is added to 1 mL of bacterial suspension.
3. Centrifuge (13000 g, 10 min, 4°C) and wash the mixture for 3 times using sterile PBS to remove excess CFDA-SE.
4. Strains are resuspended in sterile PBS (108 CFU/mL) and 1 mL of bacterial suspension is administered to SD rats. Day 1 and 3 after gavage, the fluorescence imaging is performed on anesthetized rats who are then sacrificed and different intestinal sections are taken for imaging.
5. The fluorescence imaging is performed using an IVIS Lumina III Smart Imaging System.

The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to track proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division, as well as to in vivo division of adoptively transferred cells and can resolve eight or more successive generations. CFSE is long lived, permitting analysis for several months after cell transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells. In addition, information is given on a second-generation dye, Cell Trace Violet (CTV), excited by 405-nm blue laser light. CTV is chemically related to CFSE, but allows the 488-nm line of the Argon laser to be used for other probes[2].

[1]. Quantifying CFSE Label Decay in Flow Cytometry Data. Appl Math Lett. 2013 May 1;26(5):571-577.

[2]. Flow cytometric analysis of cell division by dilution of CFSE and related dyes. Curr Protoc Cytom. 2013;Chapter 9:Unit9.11.

[3]. Prostaglandin E3 attenuates macrophage-associated inflammation and prostate tumour growth by modulating polarization. J Cell Mol Med. 2021 Jun;25(12):5586-5601.

[4]. Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment. Oncoimmunology. 2017; 6(7): e1333210.

We developed a series of models for the label decay in cell proliferation assays when the intracellular dye carboxyfluorescein succinimidyl ester (CFSE) is used as a staining agent. Data collected from two healthy patients were used to validate the models and to compare the models with the Akiake Information Criteria. The distinguishing features of multiple decay rates in the data are readily characterized and explained via time dependent decay models such as the logistic and Gompertz models.[1]

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Pancreatic ductal adenocarcinoma (PDA) remains a deadly disease that is rarely cured, despite many recent successes with immunotherapy for other malignancies. As the human disease is heavily infiltrated by effector T cells, we postulated that accurately modeling the PDA immune microenvironment would allow us to study mechanisms of immunosuppression that could be overcome for therapeutic benefit. Using viable precision-cut slices from fresh PDA, we developed an organotypic culture system for this purpose. We confirmed that cultured slices maintain their baseline morphology, surface area, and microenvironment after at least 6 d in culture, and demonstrated slice survival by MTT assay and by immunohistochemistry staining with Ki-67 and cleaved-Caspase-3 antibodies. Immune cells, including T cells (CD3+, CD8+, and FOXP3+) and macrophages (CD68+, CD163+ and HLA-DR+), as well as stromal myofibroblasts (αSMA+) were present throughout the culture period. Global profiling of the PDA proteome before and after 6 d slice culture indicated that the majority of the immunological proteins identified remain stable during the culture process. Cytotoxic effects of drug treatment (staurosporine, STS and cycloheximide, CHX) on PDA slices culture confirmed that this system can be used to assess functional response and cell survival following drug treatment in both a treatment time- and dose-dependent manner. Using multicolor immunofluorescence, we stained live slices for both cancer cells (EpCAM+) and immune cells (CD11b+ and CD8+). Finally, we confirmed that autologous CFSE-labeled splenocytes readily migrate into co-cultured tumor slices. Thus, our present study demonstrates the potential to use tumor slice cultures to study the immune microenvironment of PDA.[4]

C₂₉H₁₉NO₁₁

557.46

557.095

C, 62.48; H, 3.44; N, 2.51; O, 31.57

150347-59-4

Off-white to yellow solid powder

1.6±0.1 g/cm3

757.9±70.0 °C at 760 mmHg

152-154ºC(lit.)

412.2±35.7 °C

0.0±2.6 mmHg at 25°C

1.701

0.5

JGPOSNWWINVNFV-UHFFFAOYSA-N

InChI=1S/C29H19NO11/c1-14(31)37-17-4-7-20-23(12-17)39-24-13-18(38-15(2)32)5-8-21(24)29(20)22-11-16(3-6-19(22)28(36)40-29)27(35)41-30-25(33)9-10-26(30)34/h3-8,11-13H,9-10H2,1-2H3

(2,5-dioxopyrrolidin-1-yl) 3',6'-diacetyloxy-1-oxospiro[2-benzofuran-3,9'-xanthene]-5-carboxylate

5(6-Carboxyfluorescein diacetate succinimidyl ester; CFDA-SE; 5(6-CFDA N-succinmidyl ester;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~89.69 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.73 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)