BD Accuri C6 Plus

概述

在促進免疫學和幹細胞研究的多種應用工具中,多色流式細胞儀效果超群。免疫表型是流式細胞儀首先開發的應用之一,到現在20多年以來,BD 生物科學一直積極支援流式細胞儀系統和試劑在該領域的開創性研究.

使用直接結合有螢光抗體的試劑識別特定的細胞表面和細胞內表位,多色流式細胞計數分析允許研究者研究各個細胞在生長分化不同階段所表達的特定靶蛋白。BD提供細胞分化、轉錄因數表達和細胞因數分泌及檢測的整體解決方案,體現了對高級研究所需要的高品質和一致性的承諾.

BD Accuri™ 至尊版個人流式細胞儀將免疫學和幹細胞研究帶到了試驗臺上。它具有2個雷射器、2個散射光檢測器和載入了優化光學濾光片的4個螢光檢測器,可輕鬆處理大多數的常見試驗,同時以下2個特徵進一步簡化了實驗流程:

絕對計數.細胞濃度可直接用BD Accuri™ 至尊版的軟體統計表計算而不必加入計數微球.

試劑盒及模板. BD生物科學提供了一系列高性價比且適合BD Accuri 至尊版的免疫學和幹細胞試劑盒,包括評價多細胞表面和細胞內表型標誌物所需的試劑和緩衝液。免費的BD Accuri C6軟體範本簡化了資料獲取和分析.

下述章節和資源說明了使用BD Accuri C6進行免疫學和幹細胞應用所產生的豐富資料

資源


 

免疫分型

流式細胞儀能快速準確地免疫分型多種細胞,包括淋巴組織(胸腺、脾和淋巴結)、消化固體組織和血液等.

例如,人體血液分析的一個常見需求是識別和計算血小板、淋巴細胞、單核細胞、粒細胞和嗜酸性粒細胞的種群。流式細胞術是一種在同一時間內快速、可靠地識別單個樣品內的所有這些種群的方法,其所依據的是種群免疫分型表(見下表).

細胞亞群 免疫分型
血小板 (Platelets) CD41+CD45-
淋巴球 (Lymphocytes) CD3+CD45+
單核球 (Monocytes) CD11b+CD14+
顆粒性球 (Granulocytes) CD11b+CD14-
嗜酸性白血球 (Eosinophils) FL1++SSChigh

BD Accuri 流式細胞儀具有的若干特點使其成為人類外周血樣品差分分析的理想平臺。首先, 固定化電壓檢測器 簡化了資料收集,並減少了由於信號過度或過少擴增而導致發生資料丟失風險的可能性。第二 儀器較大的動態範圍 使得同一份資料檔案中大小不一的血小板和嗜酸性粒細胞的種群更易分析。第三 軟體縮放工具可以精確控制門的設置。最後, 直接測量體積 直接測量體積使得直接為每一種相關的種群計算其每µL細胞濃度成為可能.

資源

樣品數據

Four-color whole blood immunophenotyping panel on the BD Accuri C6 Plus
Four-color whole blood immunophenotyping panel on the BD Accuri C6 Plus
Whole blood was stained with fluorescent antibodies to CD3, CD56, CD14, and CD19 and acquired and analyzed on a BD Accuri C6 Plus. Side scatter and CD14 expression were used to discriminate lymphocyte and monocyte populations. Within the lymphocyte gate, percentages of T cells, NKT cells, NK cells, and B cells were quantified based on expression of CD3, CD56, and CD19. In all, five blood cell populations were identified in a single tube using four colors.

Identification and gating of five peripheral blood cell populations

Identification and gating of five peripheral blood cell populations
Human peripheral blood was stained and samples prepared using a red cell lyse/no-wash procedure. "Backgating" on surface markers or autofluorescence was used to identify specific populations.

Data generated on the BD Accuri C6

Using the Zoom tool to adjust gates
Using the Zoom tool to adjust gates
Human blood cells were stained and analyzed on the BD Accuri C6. A. Original polygon placement. B. Zoomed plot shows overlapping polygons. C. Adjusted polygons. D. Plot zoomed back to original scale. For method details, see the BD Biosciences technical bulletin, Identification of Human Peripheral Blood Cell Populations with the BD Accuri™ C6 Flow Cytometer (November 2011), available at bdbiosciences.com.
Differentiating naïve and memory T cells using the kit and software template
Differentiating naïve and memory T cells using the kit and software template
Human peripheral blood was stained with the BD Pharmingen Human Naïve/Memory T Cell Panel (Cat. No. 561438) according to the kit procedure, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. (The optional drop-in reagent CD3 APC-H7 was not used.) Results: A. A gate was drawn around the CD4+ T-cell population. B. The CD4+ T cells were characterized as either naïve (CD45RA+CD197+, blue), central memory (CD45RA-CD197+, red), or effector memory cells (CD45RA-CD197-, purple).
Distinguishing Tregs with CD127 using the Treg cocktail and template
Distinguishing Tregs with CD127 using the Treg cocktail and template
Human PBMCs were stained with the BD Pharmingen Human Regulatory T-Cell Cocktail (Cat. No. 560249) according to the kit procedure. The cells were then fixed, lysed and permeabilized using the BD Pharmingen™ Human Transcription Factor Buffer Set (Cat. No. 562574) and stained with PE-conjugated anti-human BD Pharmingen™ FoxP3 monoclonal antibody (Cat. No. 560082). Samples were collected on the BD Accuri C6 flow cytometer using the kit template and analyzed using BD Accuri C6 software. CD4+ lymphocytes were identified and gated by light scatter profile and fluorescence (data not shown). Results: A. A CD25 vs CD127 plot was used to identify CD25brightCD127dim Tregs and non-Treg CD4+ cells. B, C. Tregs identified using surface markers in Panel A were validated using FoxP3 staining. CD4+ cells identified as Tregs (C) expressed higher levels of FoxP3 than did non-Tregs (B).
Identifying three lymphocyte populations using two detectors
Identifying three lymphocyte populations using two detectors
Single-stained controls were used to set up gates for (A) dead cell exclusion (PI+) and (B) lymphocytes (CD45+). C. After adding antibodies to CD19, CD3, and CD16+CD56, an FL3 vs FL4 plot clearly distinguishes CD19+ B cells (purple, UL quadrant), CD56+CD16+ NK cells (magenta, LR quadrant), and CD3+ T cells (green, UR quadrant). PE-Cy5, which has high fluorescence spillover from FL3 into FL4, was used along with APC (detected primarily in FL4) and PE-Cy7 (detected primarily in FL3) to distinguish three mutually exclusive lymphocyte populations using two detectors.

Data generated on the BD Accuri C6.

 

細胞計數

Peristaltic Pumps - Image
The BD Accuri's peristaltic pumps

BD Accuri流式細胞儀獨特的液路系統用蠕動泵驅動,使得它可直接測定樣品的體積,並計數細胞。該特徵通過自動計算細胞濃度(每單位樣品體積)加速並簡化了細胞分析。準確的細胞濃度在許多研究應用中是必不可少的,包括:計數人血中的白細胞、B細胞、T細胞和血小板;測量純淨水中的微生物濃度;測定培養細胞系的活性。BD Accuri 的直接計數跟計數微球高度相關,比血球計數器的計數更準確.

可下載下面2篇關於細胞計數的BD Biosciences檔。該技術公告含有建議、技巧、技術和故障排查建議,説明將BD Accuri細胞計數和濃度的準確度最大化。該白皮書列出了樣品資料和對培養細胞系中活細胞濃度的指導、人外周血中的免疫細胞濃度和人未溶解全血中的血小板計數。

資源

樣品數據

Bacterial viability on the BD Accuri C6 Plus

Bacterial viability on the BD Accuri C6 Plus

SYBR® Green and propidium iodide (PI) were used to discriminate live vs dead E. coli bacteria after treatment with varying concentrations of ethanol. Ethanol’s bactericidal effect on cell viability was dose-dependent. Cell counts were similar using direct volume measurement in BD Accuri C6 Plus software compared to a normalized internal reference bead control.

Absolute cell counts measured by direct volume vs counting beads
Absolute cell counts measured by direct volume vs counting beads
Serial dilutions of Jurkat, 3T3, and U937 cells, and T cells, B cells, and platelet samples from four human peripheral blood donors, were counted on the BD Accuri C6 by two methods. X-axis values represent direct-volume measurements, while y-axis values were calculated based on counting beads. The direct counts correlate highly with counting beads (r2 = 0.9989) and are more precise than hemocytometer counts (data not shown).
 

細胞激素分析及基於微球的免疫分析

細胞激素和生長因子是細胞之間溝通的主要手段,它們可推動細胞分化以開發和支援免疫系統。細胞激素和生長因子的測定可以提供關於免疫反應的重要資訊,但運行多個含有單分析物的ELISA可能會花費大量的時間、勞力、預算以及樣品材料。此外,將ELISA的所有結果除以整個樣品總數,這樣可通過利用細胞的不同亞群來模糊不同細胞激素的產生差異。.

BD T-細胞的細胞激素試劑盒和試劑可簡化在BD Accuri 流式細胞儀上採用細胞內流式細胞術檢測細胞激素的過程。細胞內流式細胞術可快速分析由異構樣品內的多個表型鑒定亞群產生的細胞激素和其它炎症介質。其還可確定活化的細胞群產生細胞激素的情況是少數細胞產生了大量細胞激素還是大量細胞產生了少數細胞激素。最終,其可同時為單個細胞測定多種細胞激素.

試劑盒包括或指定了採集和分析所需的運輸抑制劑、緩衝液系統及螢光抗體。與每個試劑盒相匹配的免費 BD Accuri 軟體範本包括了預定義的工作空間、標記物、區域、門和參數名稱,以實現快速方便的安裝和分析.

當需要分析面板上廣泛分佈的細胞因數時,應考慮使用基於微球的流式細胞儀免疫測定法來分析。與ELISA測定法一樣, BD™ 流式細胞微球陣列(CBA)測定法可用於測定整個細胞群所產生的蛋白質的總量。但BD CBA可分析每個細胞因數中的300個微球—相當於300個 ELISA孔,最多可同時分析30個細胞因數,其使用的樣品非常小。從本質上講,BD CBA使用流式細胞儀等同於一次運行ELISA多達30個.

資源

樣品數據

Analysis of cytokine expression with BD CBA kits on the BD Accuri C6 Plus
Analysis of cytokine expression with BD CBA kits on the BD Accuri C6 Plus
BD CBA assays can quantify multiple cytokines simultaneously using minimal sample. In this experiment, seven cytokines were quantified in culture supernatant of stimulated PBMCs using the BD™ CBA Human Th1/Th2/Th17 Cytokine Kit. Capture beads for each cytokine were identified in FL4, and cytokine levels were measured based on bead signal intensities in FL2. Cytokine concentrations were calculated using standard curves.
Analysis of cytokine expression using intracellular flow cytometry
Analysis of cytokine expression using intracellular flow cytometry
PBMCs were stimulated with PMA + Ionomycin in the presence of BD GolgiStop™ protein transport inhibitor. The cells were fixed, permeabilized, and stained using the BD FastImmune™ Anti-Human IFN-γ FITC/IL-4 PE. Cells were acquired and analyzed on a BD Accuri C6 Plus using the kit template. IFN-γ was expressed by almost half of both CD3+ and CD3- cells, while IL-4 was predominantly expressed by CD3+ T cells.
 

血小板

有了BD Accuri 流式細胞儀,研究實驗室對血小板進行快速準確計數時便不再需要血液分析儀。.

在2001年前,血小板通常都是使用血細胞計數器或自動阻抗計數器手工計算的,儘管這些方法有很多限制。在2001年,2個專業的血液組織引入了國際參考方法(IRM)基於流式細胞術對血小板計數,儘管IRM比其他方法快速且更準確,仍然需要血液分析儀,因為大多數的流式細胞儀不能測定樣品的體積.

BD Accuri流式細胞儀獨特的液路系統用蠕動泵驅動,使得它可直接測定樣品的體積並計數細胞,這意味著它不需要血液分析儀就可計數血小板。對跨越正常範圍的血小板計數,直接體積法和IRM在BD Accuri上均經驗證為準確.

除了簡單的計數,流式細胞術在研究造成血小板活化、聚集和血栓形成個體差異較大的因素方面也是有幫助的.

資源

樣品數據

Validation of platelet counting methods on the BD Accuri C6
Validation of platelet counting methods on the BD Accuri C6

A. Correlation of platelet counts (x 109/L) obtained using the RBC/platelet ratio IRM and the direct volume method on the BD Accuri C6, and the IRM on the BD FACSCalibur™ system, for all platelet samples tested. B. Bland-Altman mean-difference analysis shows minimal bias between each pair of methods (values near zero), demonstrating that the methods are functionally interchangeable for this range of platelet samples.

In Bland-Altman analysis, the mean of the two counts for each sample is plotted on the x-axis, while their difference is plotted on the y-axis. The average difference between the two measurements is reported as the bias, with the 95% confidence interval shown in parentheses.

 

幹細胞

BD 生物科學提供了數百種針對多能幹細胞的抗體和分化標記物,可進行廣泛的選擇,它們可以許多方式組合到一起監測細胞改變的表達方式。 BD Lyoplate™ 篩查盤 可説明發現描繪感興趣細胞特徵的其他表面標誌物.

幹細胞研究的主要挑戰是細胞培養物的固有異質性。研究胚胎幹細胞(ESCs) 的研究者必須不斷地監測它們培養物的多能性,而那些研究人類誘導多能幹細胞(hiPSCs)的人必須評價重新程式設計的成功和多能性。研究、分離和培養間充質幹細胞(MSCs) 的研究者必須連續監測並驗證它們的純度和多能性.

多色流式細胞術因其同時質詢不均一細胞群體、分析它們亞群和描述多種抗原的能力,成為該用途的理想之選。每種類型的幹細胞或細胞內和表面蛋白的衍生表達特徵都可用於鑒定。由於細胞內分析需要透化作用,當研究者想分離肝細胞群體作進一步分析時,表面標記物分析是必不可少的.

資源

樣品數據

Assessing pluripotent stem cell phenotypes using surface and intracellular markers
Assessing pluripotent stem cell phenotypes using surface and intracellular markers
The BD Accuri C6 Plus was used to measure the expression of stem cell pluripotency (SSEA-4) and differentiation (SSEA-1) markers on the cell surface and pluripotency markers within the cells (Oct3/4). H9 human embryonic stem cells (WiCell) were stained with the BD Stemflow™ Human and Mouse Pluripotent Stem Cell Analysis Kit and acquired using the kit template.
Verifying the ISCT-defined MSC phenotype
Verifying the ISCT-defined MSC phenotype
Human bone marrow-derived mesenchymal stromal cells were stained using the BD Stemflow™ Human MSC Analysis Kit, acquired on a BD Accuri C6 Plus using the kit template, and analyzed for expression of MSC surface markers according to the ISCT criteria. The vast majority of analyzed cells expressed the MSC surface markers in the positive marker cocktail (CD90, CD105, and CD73, B–D), while very few expressed those in the negative marker cocktail (CD34, CD11b, CD19, CD45, and HLA-DR, A). Gates were drawn based on matched isotype control cocktails (not shown).



All reagents and kits are compatible with both the BD Accuri C6 Plus and BD Accuri C6 flow cytometer systems. Platforms referred to as "BD Accuri" represent both the BD Accuri C6 Plus and BD Accuri C6. Data was generated on either the BD Accuri C6 Plus or the BD Accuri C6 as indicated in figure legends.