Permeabilization and fixation conditions for intracellular flow cytometric detection of the T-cell receptor ? chain and other intracellular proteins in lymphocyte subpopulations

Lazarus, Alan H.; Ellis, Janet; Blanchette, Victor S.; Freedman, John C.; Sheng-Tanner, Xiofang

doi:10.1002/(sici)1097-0320(19980701)32:3<206::aid-cyto7>3.0.co;2-h

Cited by 14 publications

(8 citation statements)

References 30 publications

(25 reference statements)

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“…After 3 washings, fluorescence activity was detected from cell fractions containing 5000 cells using a FACS caliber at 480 nm (CD11b) or 580 nm (DHE), and the data were analyzed with CellQuest software (Becton Dickinson). When fluorescence activity was detected, data was obtained for granulocytes and monocytes using gating definitions as previously reported [18]–[19].…”

Section: Methodsmentioning

confidence: 99%

“…After 3 washings, fluorescence activity was detected from 5000 cell fractions using a FACS caliber and the data were analyzed with CellQuest software (Becton Dickinson). When fluorescence activity was detected, data were obtained from monocyte/macrophage and dendritic cells (DCs) subsets using gating definitions as previously reported [14], [18]–[20]. The cell population in SVF consisted of M1 (F4/80 + /CD11c + ) and M2 (F4/80 + /CD204 + ) macrophages, activated monocytes (CD11b + /CD11c + ), and 2 subpopulations of mature dendritic cells (DCs) (CD11c + /CCR7 + , CD11c + /CD86 + ), as previously reported [5], [8], [14], [21]–[27].…”

Section: Methodsmentioning

confidence: 99%

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Adipose Inflammation Initiates Recruitment of Leukocytes to Mouse Femoral Artery: Role of Adipo-Vascular Axis in Chronic Inflammation

et al. 2011

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BackgroundAlthough inflammation within adipose tissues is known to play a role in metabolic syndrome, the causative connection between inflamed adipose tissue and atherosclerosis is not fully understood. In the present study, we examined the direct effects of adipose tissue on macro-vascular inflammation using intravital microscopic analysis of the femoral artery after adipose tissue transplantation.Methods and ResultsWe obtained subcutaneous (SQ) and visceral (VIS) adipose tissues from C57BL/6 mice fed normal chow (NC) or a high fat diet (HF), then transplanted the tissues into the perivascular area of the femoral artery of recipient C57/BL6 mice. Quantitative intravital microscopic analysis revealed an increase in adherent leukocytes after adipose tissue transplantation, with VIS found to induce significantly more leukocyte accumulation as compared to SQ. Moreover, adipose tissues from HF fed mice showed significantly more adhesion to the femoral artery. Simultaneous flow cytometry demonstrated upregulation of CD11b on peripheral granulocyte and monocytes after adipose tissue transplantation. We also observed dominant expressions of the inflammatory cytokine IL-6, and chemokines MCP-1 and MIP-1β in the stromal vascular fraction (SVF) of these adipose tissues as well as sera of recipient mice after transplantation. Finally, massive accumulations of pro-inflammatory and dendritic cells were detected in mice with VIS transplantation as compared to SQ, as well as in HF mice as compared to those fed NC.ConclusionOur in vivo findings indicate that adipose tissue stimulates leukocyte accumulation in the femoral artery. The underlying mechanisms involve upregulation of CD11b in leukocytes, induction of cytokines and chemokines, and accumulation of pro-inflammatory cells in the SVF.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Adipose Inflammation Initiates Recruitment of Leukocytes to Mouse Femoral Artery: Role of Adipo-Vascular Axis in Chronic Inflammation

et al. 2011

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“…Cluster of differentiation (CD)3z is a key protein involved in T-cell receptormediated signal transduction, 9,10 and down-modulation of this protein causes a reduction of the cytotoxic effector function and production of T-helper (Th)-1 cytokines, and proliferative responses in T cells following antigenic or mitogenic stimulation. 11,12 The down-modulation of CD3z in T cells has been described in various human tumors, [11][12][13][14][15][16][17][18][19] human immunodeficiency virus (HIV) infection, 20 and autoimmune disease. 10,21 Previous studies attempted to explain the significance and mechanism of the down-modulation of CD3z from the viewpoints of the interaction of T cells with activated macrophages 22,23 and the tumor-inducing apoptosis of T cells.…”

Section: Introductionmentioning

confidence: 99%

Decreased expression of CD28 coincides with the down‐modulation of CD3ζ and augmentation of caspase‐3 activity in T cells from hepatocellular carcinoma‐bearing patients and hepatitis C virus‐infected patients

Maki

Matsuda

Asakawa

et al. 2004

J of Gastro and Hepatol

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“…Second, as this study shows, digitonin can reduce the total staining protocol to less than 10 min which is important in a high frequency, real time analysis. Fourth, the general strategy of using digitonin to rapidly permeabilize cells can potentially be applied to other stains or fluorescently labeled antibodies to quantify intracellular targets (32–34).…”

Section: Discussionmentioning

confidence: 99%

Growth dynamics of mammalian cells monitored with automated cell cycle staining and flow cytometry

Sitton

Srienc

2008

Cytometry Pt A

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To accurately observe high-frequency events during transient cell cycle kinetics, we have implemented a single step 15-min DNA staining protocol using automated flow cytometry. This protocol was used to sample and to analyze a Chinese hamster ovary cell culture for the DNA distribution, viable cell concentration, apoptotic cell concentration, and light scattering properties every 25 min over 4.5 days in response to a nutrient deprivation and a nutrient upshift. After the nutrient deprivation and exposure to fresh growth medium, two populations of cells started proliferating at different times likely corresponding to cells leaving the G0 and G1 cell cycle phases. After a nutrient upshift in late exponential growth, a cell cycle arrest occurred at the G1/S and G2/M boundary. The resulting cell cycle and proliferation kinetics followed damped oscillations that directly reveal the average time cells spend in each cell cycle phase. The observed detailed dynamics of the cell cycle progression is made possible through the high-frequency sampling enabled by automated flow cytometry. The approach should be useful in studying cell cycle perturbations in response to different environmental conditions resulting from exposure to specific nutrients or to drugs. ' 2008 International Society for Advancement of CytometryKey terms automated flow cytometry; population dynamics; cell cycle staining; digitonin; mammalian cell culture; single cell heterogeneity DETERMINATIONof the cell cycle distribution is one of the most basic and most widely used applications of flow cytometry in basic biological research and in diagnostic applications. It traditionally involves cell fixation and permeabilization and cell staining with nucleic acid specific dyes, often after enzymatic removal of RNA. Variations of this methodology have been well documented in many reports, review articles and textbooks (1). The cell preparation and staining steps typically involve several manual operations, and their careful execution determines the reproducibility and the quality of the cell cycle distribution that can be obtained. However, in many cases a large fraction of cells may be lost during fixation and staining steps which introduces the additional problem that the remaining cells may not be representative of the entire sample.Traditional cell cycle staining protocols use either cell membrane permeable or impermeable dyes. Staining protocols using membrane permeable dyes are typically simple and short involving only addition of the dye to the cells. However, membrane permeable dyes often are more expensive in comparison to membrane impermeable dyes [Vybrant, DRAQ5 (2)] or limited to a UV excitation source (Hoechst, DAPI). In contrast, membrane impermeable dyes such as PI or Acridine Orange are inexpensive and can be excited by common 488 nm light sources. Cell cycle staining protocols using membrane impermeable dyes are usually long, multistep procedures.Time dependent changes in the cell cycle distribution of a growing cell population reflect t...

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Permeabilization and fixation conditions for intracellular flow cytometric detection of the T-cell receptor ? chain and other intracellular proteins in lymphocyte subpopulations

Cited by 14 publications

References 30 publications

Adipose Inflammation Initiates Recruitment of Leukocytes to Mouse Femoral Artery: Role of Adipo-Vascular Axis in Chronic Inflammation

Adipose Inflammation Initiates Recruitment of Leukocytes to Mouse Femoral Artery: Role of Adipo-Vascular Axis in Chronic Inflammation

Decreased expression of CD28 coincides with the down‐modulation of CD3ζ and augmentation of caspase‐3 activity in T cells from hepatocellular carcinoma‐bearing patients and hepatitis C virus‐infected patients

Growth dynamics of mammalian cells monitored with automated cell cycle staining and flow cytometry

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