Flow Cytometry流式細胞儀設施

Brand

Q & A


  • Analyzers

Q1. What notice should I take of before reserving analyzers?

Q2. What kind of tube should be used for sample injection?

Q3. What concentration of samples is suggested for sample preparation?

Q4. Is a negative control necessary for every experiment?

Q5. Are single stained controls essential for multi-color experiments?

 


  • Cell sorters

Q1. How can I reserve my sorting time?

Q2. Can I cancel my reserved time?

Q3. How many cells do I need to bring? How long should I book for my cell sorting?

Q4. How many populations can I sort simultaneously?

Q5. How to prepare my cells for sorting and at what concentration?

Q6. What kind of tubes should be prepared for sample injection and collection?

 

 

  • Analyzers

​Q1. What notice should I take of before reserving analyzers?

Please make sure that the analyzer you reserved was equipped the laser with correct excitation wavelength.  For user of first time coming, please contact instrument manager first to reserve hands-on course.

 

Q2. What kind of tube should be used for sample injection?

If you use the FACSCanto and LSRII analyzers, please prepare samples in 5mL Falcon polystyrene tube (Cat. 352052, 352054, 352235); if you use the Attune NxT analyzer, samples can be prepared in 1.5-2mL Eppendorf, microtube or any 5mL tube.

 

Q3. What concentration of samples is suggested for sample preparation?

If you use the FACSCanto and LSRII analyzers, we suggest you to use samples with concentration of 1~2x106 cells/mL; if you use the Attune NxT analyzer, we suggest you to use samples with concentration of 0.5~1x106 cells/mL.

 

Q4. Is a negative control necessary for every experiment?

Yes. Due to sample and reagent condition and optical adjustment of the analyzer will be different from every time, we recommend that a negative control or isotype control is necessary in every experiment for correct adjustment of instrument setting and accurate definition of positive signal. If you still have any questions of experimental designs, please feel free to contact the instrument manager.

 

Q5. Are single stained controls essential for multi-color experiments?

Yes. When more than two kinds of fluorescence are detected simultaneously, spectra overlap and fluorescence spillover will be generated and cause to false positive results. To prevent both phenomena, user should prepare single stains for fluorescence compensation adjustment. Furthermore, fluorescence spillover will affected by preparation or conservation of samples and reagents, so we highly recommend that single stain controls are essential every time. If you still have any questions of experimental design, please feel free to contact the instrument manager.

 


  • Cell sorters

Q1. How can I reserve my sorting time?

Please contact the sorter operator (2652-3927 or 2652-3036) to reserve the sorting time.

 

Q2. Can I cancel my reserved time?

Yes.  You should cancel your reserved time at least one day earlier by contacting the sorter operator.  Without cancellation or cancellation on that day will still be charged full amount of the reservation time.

 

Q3. How many cells do I need to bring? How long should I book for my cell sorting?

This will depend on how many cells you want to recover. For example, if the target cells you are interested in sorting are 10% of your sample and you want to recover 1 x 106 target cells, you need to prepare 2 x 107 as a starting cell number (2 x 107 x 10% x 50% = 106). It is always best to calculate the number of input cells based on the worst-case scenario of a 50% yield rate so you will be sure to have an ample number of cells to start. Generally speaking, 2 x 107 cells will take 1 hour for sorting. 

 

Q4. How many populations can I sort simultaneously?

Up to four cell populations can be sorted simultaneously by FACSAria, and two cell populations can be sorted simultaneously by FACSJazz.

 

Q5. How to prepare my cells for sorting and at what concentration?

The samples for sorting must be in a single cell suspension and filtered by nylon mesh (mesh size: 35µm) before sorting to prevent clogging. The sorting buffer can be PBS or HBSS containing 1%~2% FBS and 25mM HEPES. For sticky samples or samples with high percentage of death cells, you can add 1~5mM EDTA or DNAse in sorting buffer. We recommend a concentration of 5~10x106 cells/mL for sample preparation.

 

Q6. What kind of tubes should be prepared for sample injection and collection?

For sample injection: If using the FACSJazz sorter, sample can only be prepared in FALCON 5mL polypropylene round-bottom tube (Cat. #352063); if using the FACSAria sorter, sample can be prepared in any 5mL (12x75mm) round-bottom tube or 15mL centrifuge tube.

For sample collection: Both two sorters can sort cells into 5mL (12x75mm) round-bottom tube, 15mL centrifuge tube, 6-384 well plates or onto slide.

 


 

常見問題


  • 分析儀

Q1. 預約機器前該注意哪些事項?

Q2. 樣本需放在何種上樣管中?

Q3. 建議的樣本濃度?

Q4. 是否每次都需準備negative control?

Q5. 分析多色螢光實驗時,是否每次都需要準備單染樣本?

 


  • 分選儀

Q1. 如何預約細胞分選服務?

Q2. 可否取消已預約的分選時段?

Q3. 需要準備多少細胞數目進行細胞分選?需要預約多少分選時段?

Q4. 一次細胞分選實驗中可以同時收集幾群細胞?

Q5. 如何準備細胞分選樣品?適當的細胞濃度為何?

Q6. 要準備何種上樣管及收集管?

 


  • 分析儀

Q1. 預約機器前該注意哪些事項?

請先確認該機器有搭載合適的激發波長的雷射。第一次上機,請先行與管理者預約教學時間。

 

Q2. 樣本需放在何種上樣管中?

若您是使用FACSCanto或LSRII分析儀,請將樣本置於5mL Falcon polystyrene tube (Cat. 352052, 352054, 352235);若您是使用Attune NxT分析儀,可將樣本置於1.5-2mL eppendorf、microtube或任何5mL tube。

 

Q3. 建議的樣本濃度?

若您是使用FACSCanto或LSRII分析儀,建議樣本濃度為1~2x106 cells/mL;若您是使用Attune NxT分析儀,建議樣本濃度為0.5~1 x106 cells/mL。

 

Q4. 是否每次都需準備negative control?

是的。由於每次實驗樣本、試劑狀態及儀器光學校準都可能有差異,為了能正確調整儀器設定,並且在分析樣本時可精確定義陽性(positive)螢光訊號範圍,我們建議每次實驗都需要準備negative control或isotype control。若您仍有任何相關實驗設計問題,歡迎與儀器管理員討論。

 

Q5. 分析多色螢光實驗時,是否每次都需要準備單染樣本?

是的。兩種以上螢光在分析儀中被同時偵測時,可能存在光譜重疊(spectra overlap)或螢光溢散(fluorescence spillover)問題而造成偽陽性(false positive)結果,為解決此現象,必須以單染樣本進行螢光補償(compensation)進行校正,如此才能得到正確的螢光訊號。又螢光溢散現象可能會因樣本或試劑的處理或保存狀態而改變,因此我們建議每次實驗都需要準備單染樣本。若您仍有任何相關實驗設計問題,歡迎與儀器管理員討論。

 


  • 分選儀

Q1. 如何預約細胞分選服務?

請撥打電話(2652-3927或2652-3036)與分選儀操作人員預約時間。

 

Q2. 可否取消已預約的分選時段?

可以。最晚須於預約日的前一天向操作人員取消預約。未取消或當日取消者,仍將按預約時段收取費用。

 

Q3. 需要準備多少細胞數目進行細胞分選?需要預約多少分選時段?

沒有限制使用者準備的細胞數目,使用者可以先估算自己實驗所需的細胞數目,例如預計要分選的目標細胞群佔總細胞的10%,使用者想要回收的細胞數目為106顆細胞時,請準備2x107顆細胞來進行細胞分選,算式為2x107x10%x50%=106 (50%為進行細胞分選時預估至少有一半的回收率)。依據使用者所準備來的細胞數目,來預約細胞分選所需的時間,以2x107顆細胞來說,需要預約1小時的分選時段,以確保能完成實驗。目前本設施分選時段是以半小時(30分鐘)為一個單位,最少需預約半小時。

 

Q4. 一次細胞分選實驗中可以同時收集幾群細胞?

如果是預約FACSAria的話,最多可以同時收集4群細胞;若是預約FACSJazz的話,最多可以同時收集2群細胞。

 

Q5. 如何準備細胞分選樣品?適當的細胞濃度為何?

使用者需要先將細胞樣品處理成單顆細胞懸浮液,回溶於PBS或HBSS中,內含1~2%FBS及20mM HEPES。若是樣品有特殊情況,如細胞容易聚集或是包含較多的死亡細胞,可以額外添加EDTA、DNAse或是任何適合細胞分選的緩衝液中。上機前要先將樣品過濾,避免塞機。上機細胞濃度建議調整為5~10x106顆細胞/mL。

 

Q6. 要準備何種上樣管及收集管?

若是預約FACSJazz進行細胞分選實驗,請將上機樣品準備在 FALCON 5mL polypropylene round-bottom tube (Cat. #352063)中。而FACSAria則可以使用任何 5mL (12x75mm)圓底管或是15mL離心管,並無限制材質及廠牌。兩台分選儀皆可使用任何 5mL (12x75mm)圓底管裝1mL培養液或是15mL離心管裝3mL培養液做為樣品收集管,無限制材質及廠牌,亦可將細胞分選於多孔盤中。

 


 

Top