Cfse dilution

Manufactured by Thermo Fisher Scientific

CFSE dilution is a fluorescence-based technique used to monitor cell division and proliferation. It involves the labeling of cells with a fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE), which binds to intracellular proteins. As cells divide, the CFSE dye is partitioned equally between daughter cells, resulting in a progressive halving of the fluorescence intensity with each cell division. This allows for the quantification of the number of cell divisions that have occurred.

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Lab products found in correlation

Foxp3 fix perm kit, by Thermo Fisher Scientific (1 mentions) Cd138 pe, by BD (1 mentions) Diva software, by BD (1 mentions) Lsr 2, by BD (1 mentions) Cd45.2 pe, by BD (1 mentions) Irf4 pe, by Thermo Fisher Scientific (1 mentions) Irf8 percp efluor710, by Thermo Fisher Scientific (1 mentions) Cd45.1 apc, by BD (1 mentions) Negative selection, by STEMCELL (1 mentions) Mitomycin, by Merck Group (1 mentions)

4 protocols using cfse dilution

Comprehensive B Cell Immunophenotyping

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B cell subsets were identified with CD22.2-FITC, CD138-PE, CD45.2-PE, CD45.1-APC (BD Biosciences), CD45R/B220-eFluor450, CD44-eFluor780 (eBioscience). RF B cells were detected with biotinylated-4G7 in combination with streptavidin-PerCP-Cy5.5. IRF-4 was detected using an IRF-4 antibody (clone M-17, Santa Cruz) and anti-goat IgG Alexa Fluor 647 (Jackson ImmunoResearch). IRF4-PE and IRF8-PercP-efluor710 (eBioscience) were used to co-stain IRF8 and IRF4. B cell proliferation was assessed by CFSE dilution (Life Technologies) (15 ) or VPD450 dilution (BD). Dead cells were distinguished with TO-PRO-3 (Life Technologies). To analyze TLR7 expression levels, unstimulated purified B cells, or B cells stimulated for 24 hr, were fixed and permeabilized using the Foxp3 Fix/Perm kit (ebioscience). TLR7 protein was detected using a biotinylated mouse TLR7 specific mAb, A94 (25 (link)), in combination with SA-PE. Flow cytometric analysis was carried out using a BD LSR II with Diva Software (BD) and analysis was conducted with FlowJo software (Tree Star).

Nündel K., Green N.M., Shaffer A.L., Moody K.S., Busto P., Eilat D., Miyake K., Oropallo M.A., Cancro M.P, & Marshak-Rothstein A. (2015). Cell intrinsic expression of TLR9 in autoreactive B cells constrains BCR/TLR7-dependent responses. Journal of immunology (Baltimore, Md. : 1950), 194(6), 2504-2512.

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Evaluating T Cell Homeostatic Proliferation

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T cell homeostatic proliferation in vivo were determined in CD45.2 HUPO or control mice or HUPO or littermate recipients on Rag-/- background. The CD3⁺ T cells were purified from CD45.1 mice by negative selection (StemCell Technologies) and CFSE labelled. Adoptive cell transfers were performed by intravenous administration into CD45.2⁺ cells (1x10⁷/200 μl) into recipients. Three to 6 days after transfer, spleens were harvested and T cells analyzed by flow cytometry for homeostatic proliferation by CFSE dilution (Life Technologies),²⁷ (link)^,⁷⁸ (link) and to identify Tregs and Treg Foxp3 expression.

Huang Q.Q., Hang Y., Doyle R., Mao Q., Fang D, & Pope R.M. (2023). Mechanisms regulating the loss of Tregs in HUPO mice that develop spontaneous inflammatory arthritis. iScience, 26(5), 106734.

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Isolation and Characterization of MDSCs and DCs

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MDSC were isolated from splenocytes by positive selection with FITC anti-Ly6C, PE anti-Ly6G, or PE anti-Gr1 using anti-FITC or anti-PE microbeads (Miltenyi Biotec), as described (12 , 14 (link)). DC were isolated by CD11c immunomagnetic bead selection and γ-irradiated (20 Gy). T cell proliferation was assessed at 72 h by [³H] TdR incorporation or CFSE dilution (Invitrogen).

Rosborough B.R., Mathews L.R., Matta B.M., Liu Q., Raïch-Regué D., Thomson A.W, & Turnquist H.R. (2014). Flt3 ligand mediates STAT3-independent expansion, but STAT3-dependent activation of myeloid-derived suppressor cells. Journal of immunology (Baltimore, Md. : 1950), 192(8), 3470-3473.

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Proliferation of CD4+ T Cells

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To determine the proliferation of CD4⁺ T cells, splenocytes (collected by day 8) were stained with CFSE dilution (Invitrogen). Thereafter, total splenocytes were incubated with stimulator DBA/2 splenocytes treated with mitomycin (Sigma-Aldrich). After 5 days of culture, cells were analyzed by flow cytometry.

Maenosono R., Nian Y., Iske J., Liu Y., Minami K., Rommel T., Martin F., Abdi R., Azuma H., Rosner B.A., Zhou H., Milford E., Elkhal A, & Tullius S.G. (2021). Recipient Sex and Estradiol levels affect transplant outcomes in an age-specific fashion. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 21(10), 3239-3255.

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