Cell Function
Cell Function FAQs
In order to provide better customer support and answer customers' questions about cell function assay products, we have compiled and published answers to the frequently-asked questions (FAQs) about cell function assay products, and will continue to update them.
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A 50 assays kit can detect 50 samples with the size of about 5cm2 (the size of a cover slide or 12-well plate) without positive or negative control. It will take another two assays for control groups.
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ElabFluor®488 offers better quenching resistance and is more suitable for longer microscopic observations than that of FITC.
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Color development of DAB (result of TUNEL-HRP) is observed with an ordinary microscope, the results of TUNEL nuclear staining were served with white light. As for TUNEL fluorescence, the results of TUNEL and nuclear staining were observed with different fluorescence channels seperately with better definition and resolution.
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1. To avoid the effect of light on TdT enzyme activity.
2. Biotin molecules are easily decomposed by light, and the double bonds in biotin molecules are easily excited by light, and the decomposition produces substances such as ultraviolet light and active free radicals. These decomposition products will destroy the chemical binding between biotin and labeled molecules, thus affecting the accuracy and stability of experimental results. -
Yes they do. The blocking agent can be 3% H2O2. Peroxidase is present in many tissues, resulting in false positive results. Thus the endogenous peroxidase should be eliminated before staining. 3% hydrogen peroxide can react with peroxidase to eliminate endogenous peroxidase as a blocking agent.
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There is no species limination in TUNEL assay kits.
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Yes, it can be used to test sperm samples.
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There is no species limination in TUNEL assay kits, so it will be avaliable theromatically. It is recommanded for customers to explore the method of sample preparation as there is no such data for insect samples.
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The detection of TUNEL is based on DNA fragmentation in late stage apoptosis, so it is not avaliable for erythrocyte as there is no nuclei or other organelles containing DNA in erythrocyte.
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(1) The slides can be sealed with anti-fluorescence quenching agent, but the microplate cannot.
(2) The amount of reagents will be increased with 6-well plate.
(3) The visual field will be decreased due to the smaller diameter of wells. -
It is avaliable therometically, but it is recommanded to take a pre-test as there is no such data yet. You might need to notice some key points: 1. Protease K for section sample treatment as permeability agent may interfere the antigen of immunofluorescence; 2. High temperature or high pressure for antigen repair in immunofluorescence assay may lead to DNA fragmentation, that will end up with false positive TUNEL results.
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Yes, the cells can be collected into EP tubes as cell suspension. After being stained by following the procedure, the cells can be detected by flow cyrometer, or the suspension can be sucked onto a slide and observed with a microscope.
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No, it doesn't. What's more, antigen repair may cause DNA fragmentation and lead to false positive signal.
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Do not dry frozen section. Frozen section can be stored in -20°C within a week or in -80°C for longer time after being prepared. Before the experiment, rewarm the section at room temperature for about 20 minutes, fix at room temperature for 30-60 minutes with 4% of paraformaldehyde formaldehyde, then wash it and follow the procedure on the manual.
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The TdT equilibration buffer in the TUNEL kit is designed to provide optimal reaction conditions for the TdT enzyme.
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It is recommended to dilute with ultra-pure water, DO NOT use DEPC water. DEPC water is used to protect RNA or DNA from enzymatic hydrolysis, it will deactive Dnase I. DEPC water can be used to dissolve RNA when extracting RNA.
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Yes, soak in PBS and store at 4℃.
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If it has been fixed, it is not necessary to incubate and fix again, and subsequent experimental steps can be directly carried out.
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DO NOT use neutral balsam. The section is in a water-soluble state after dyeing, while neutral balsam is a water-insoluble agent that will blurred the sample. It is recommended to use a water-soluble sealer.
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Yes, it is. If DAPI is already contained in the anti-fluorescence quenching agent, there is no need to stain the sample with extra DAPI in the kit.
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No. TUNEL works by attaching a fluorescently labeled dUTP to the 3'-OH terminal of the fragmented DNA, it can only be worked once since there is no 3'-OH terminal after the operation.
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Cells with high proliferative or metabolic active may have a break in the DNA strand, both cases may end up with false-positive result. Evaluation of cell morphology may be used if there are doubts about the interpretation of the results.
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The tissue is transparent, and no water drop can be left on the slide.
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The main difference lies in the detection methods, for TUNEL in-situ kits, a fluorescence microscope is used for observing the results, as for TUNEL flow ytometry kit, the sample will be detected by a flow cytometer.
On the other hand, TUNEL in-situ kit is more suitable for tissue sections and cell slides or smears, while TUNEL flow ytometry kit is more suitable for suspended cells and adherent cells. -
It is not recommended to do so. Platelet and some other immune cells in blood may have exposed PS in normal state that will affect the test results.
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Annexin V kit can be used on adherent cells, with the following caveat:
1. In late stage of apoptosis, cells may not able wo maintain adhesion, so floating cells should be collected for the test.
2. The dissociation of adherent cells should be handled gently to avoid cell damage caused by human operation.
3. Do not digest for too long with trypsin, avoid the usage of the trypsin contain EDTA. If EDTA-containing trypsin must be used, wash the cells to remove EDTA as much as possible. -
If there is a DAPI channel in the flow cytometry, the E-CK-A258 Annexin V-APC/DAPI Apoptosis Detection Kit will be suitable. If not, the E-CK-A218 Annexin V-APC/7-AAD Apoptosis Detection Kit is avaliable.
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There is no such experience as verification yet. It is recommanded to take a pre-test if necessary.
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It is not recommended to do so. Cryopreservation may lead to apoptosis and necrosis, resulting in inaccurate experimental results.
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Annexin V-Elab Fluor® Red 780/DAPI Apoptosis Kit E-CK-A280 will be the best choice with minimun interference.
However, if there is no DAPI channel, Annexin V-Elab Fluor® Red 780/7-AAD Apoptosis Kit E-CK-A240 may also be avaliable.
Elab Fluor® Red 780 was detected by APC-CY7 channel. If there is neigher DAPI channel nor APC-CY7 channel, Annexin V-PE/7-AAD Apoptosis Kit E-CK-A216 could be a choice but with more interference. -
Annexin V-APC/DAPI Apoptosis Kit E-CK-A258 is available, but requires a DAPI channel.
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No, because Annexin V binds to phosphatidylserine (PS) dependent on Ca2+, which is not present in common PBS solutions, while Binding buffers contain Ca2+ to facilitate Annexin V binding to PS. Without Binding Buffer, Annexin V binding capacity will be reduced, resulting in significantly reduced positive signal or even no positive signal.
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Annexin V assay is normally used for cell line samples, thus only debris need to be excluded by gating. Since there aremultiple types of cells in tissue samples, it depends on the purpose of experiment, whether it means to detect apoptosis of the whole tissue sample or some specific cells. Cells can be gated by FSC/SSC or some other biomarkers.
Besides, operation of tissue sample preparation may also effect the result, Inappropriate operation may also result in false positives due to sample handling. -
The purpose of fixing cells is to preserve the original structure of tissues and cells as complete as possible, avoid degradation, autolysis, corruption, and deformation of tissues and cells, deactive various enzymes in cells and tissues to prevent various molecular degeneration and dissociation, and enable cell chemicals and enzymes to be accurately located. And in the future processing and production process will not change and damage. At the same time, fixation can also make each part of the cell easy to be stained, suitable for observation, long-term preservation and analysis.
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Absolute ethanol is anhydrous ethanol which is 100%. It is not recommended to be replaced by 70% ethanol in order to match the best condition of assay.
If there is a sub-G1 spike, it can be detected. Due to nucleus concentration and DNA fragmentation, some genomic DNA fragments of apoptotic cells were lost during the staining process. Therefore, apoptotic cells showed significantlly weaker staining after PI staining than that of the normal cells, that is, the fluorescence intensity was less than 1. The so-called sub-G1 peak, or apoptotic cell peak, appeared on the flow detection result map. -
Serum-free medium is necessary for cell cycle assays. Before the start of the experiment, cells should be cultured in serum-free medium for 12h to synchronize the cycle (pay attention to cell status, do not culture cells without serum for too long, or the cell state may be too poor to proliferate, end up with shrunk G2 phase or even sub-G1 apoptotic peak). Then normal medium was used for drug treatment, intervention and other experiments, and finally samples were collected for cell cycle detection.
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Flow cytometry: 125 samples (1~5×10^5 cells)
Fluorescence microscopy: 100 samples (96-well plate), 50 samples (24-well plate) -
Calcein AM has strong fluorescence only after being hydrolyzed by endogenous esterase in the cell. Free calcein AM in buffer is almost non-fluorescent, so it does not need to be washed before detecting.
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It is not recommended to dilute the dye before repackage, since it may lead to hydrolysis of Calcein AM as Calcein AM is more stable in stock solution. Calcein AM stock solution can be divided according to needs, and can be stored at -80°C for a longer shelf life. It is better to prepare the diluted buffer right before use, and use it up within one day.
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The minimum number of cells is 1 million, and the protein concentration is between 1 and 4 mg/mL.
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sf9 cells are insect cells, it can be used if caspase is indeed involved in the apoptosis pathway of insect cells.
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The Caspase 9 kit measures the enzyme activity of caspase 9, which is activated caspase 9, also known as dimers or a tetramers of cleaved caspase 9.
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JC-1 needs to detect the changes of fluorescence signal in both Ex/Em = 514/529 and Ex/Em = 585/590 at the same time to determine the changes of mitochondrial membrane potential, and there is fluorescence interference with GFP, so this kit cannot be applied to cells with GFP.
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JC-1 exists in monomer and polymer form, and their emission spectra are different. In normal cells with high mitochondrial membrane potentia, JC-1 accumulates in the matrix of mitochondria as polymer, which producing red fluorescence. Since the decreasing of mitochondrial membrane potential during early stage of apoptosis, JC-1 can no longer be accumulated in the mitochondrial matrix, that becomes monomer with green fluorescence. Therefore, both red and green fluorescence need to be observed.
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It is not recommended to do so. Cells may suffer apoptosis or damage due to mechanical manipulation during preparation into cell suspension, in which case the results of JC-1 may be inaccurate. In addition, this kit can only detect live single-cell suspensions and cannot be used to slice samples.
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MTT assay kits are widely used to detect cell proliferation and cytotoxicity, and MTS, XXT, WST-1 and WST-8 are all upgraded alternatives to MTT, which have obvious advantages compared with MTT. On the one hand, the reaction production of MTT assay is insoluable in water, a specific solvent is required in the assay, products of WST-8, XTT and MTS are water-soluble with easier operation. On the other hand, the solubility of the formazan produced of WST-8 is higher than that of XTT and MTS. What's more, WST-8 is more stable than MTT, XTT, and has a wider linear range and higher sensitivity.
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The effect of drug color can be eliminated by changing the medium after the drug incubation and before adding CCK-8.
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BrdU method has many steps and requires the use of BrdU antibody, which has many influencing factors and poor stability. Based on EdU incorporation and subsequent click reaction, EdU method does not need to use antibodies, it has convenient operation and high detection sensitivity. It is a new method based on BrdU method, which will gradually replace BrdU method. EdU (5-ethynyl-2-deoxyuridine) is a thymidine analogue that can be incorporated into the newly synthesized DNA instead of thymidine in the process of DNA synthesis. The acetylene group on EdU can covalently react with fluorescence-labeled small molecular azide probes (such as FITC Azide, ElabFluor ®488 Azide, ElabFluor ®594 Azide, ElabFluor ®647 Azide) to form a stable triazole ring catalyzed by univalent copper ions, this reaction is called click reaction (Click reaction).
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No. Elab Fluor® 594-11-dUTP is a single component, while Labeling Solution is a multi-component mixed reagent that includes labeled dUTP. Therefore, it is recommended that customers purchase TUNEL kits directly for experiments.
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① The test sample does not contain fluorescence, and all TUNEL kits can be used. ② If the experimental sample has red light, it is recommended to use One-step TUNEL Assay Kit (Green, FITC) item No. : E-CK-A320 or One-step TUNEL Assay Kit (Green, Elab Fluor®488) item No. : E-CK-A320.
In general, the anti-quenching effect of Elab Fluor®488 is better than that of FITC, and One-step TUNEL Assay Kit (Green, Elab Fluor®488) is recommended if the green fluorescence of a sample is to be observed over a long period of time.
③ If the experimental sample has green light, One-step TUNEL Assay Kit (Red, Elab Fluor®594) is recommended: E-CK-A322 or One-step TUNEL Assay Kit (Red, Elab Fluor®555) Item No. E-CK-A325. The Elab Fluor®594 Assay Kit (Red, Elab Fluor®594), item No. E-CK-A322, is preferred if the sample has a strong green fluorescence.
If both Red and green fluorescence are present in the test sample, One-step TUNEL Assay Kit (Red, Elab Fluor®647) is recommended: E-CK-A324, but Elab Fluor®647 has higher requirements for fluorescence microscopy, requiring the microscope to be equipped with Cy5 or Elab Fluor®647 filters, which are generally equipped with confocal microscopes. -
It is recommended to incubate 500μL of Labeling Working Solution per well.
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The negative control group needs to perform labeling protocol too. However, it should be noted that the Labeling Working Solution in the negative control group are different from the other positive control or experimental group.
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It requires DAPI working Solution to cover the sample.
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It can be washed and soaked in PBS and stored in a refrigerator at 4°C.
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Yes, the frozen samples of -80°C can be made into frozen slices, and then the frozen slices can be used for tunel detection.
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Generally, the thickness of frozen slices is 8-10 µm. If the slice is thick, the incubation time of protease K can be extended appropriately according to the actual situation.
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The process of making paraffin sections requires baking sheets, but the process of tunel dyeing after the section is made does not require baking sheets.
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PBS added during washing steps must completely cover the sample, and ensure the sample moist during the experiment to prevent the failure of the experiment caused by dry slides.
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It is recommended to incubate at room temperature, about 25℃. The AnnexinV staining process is actually the process of binding the AnnexinV protein specifically to phosphatidylserine on the cell membrane, and this process is appropriate when the cell function and enzyme activity are normal.
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According to the specifications of the kit and the method of use, the specific number of samples can be determined. There are two ways to use annexin V in the instructions, one step method and two step method. The one-step annexin V dosage is 5μL, the two-step annexin V dosage is 2.5μL, and we are provided in accordance with the one-step method (maximum dosage), which means that if you do it in two steps, the kit size of 20 Assays can do 40 samples, and so on.
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Check to see if your device has a DAPI channel and recommend the Annexin V-APC/DAPI Apoptosis Kit, item number E-CK-A258.
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No, the concentration of calcium ions in binding buffer is determined after experimental exploration, and PBS containing calcium ions may not have the effect of binding buffer.
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It is not advised to use tissue samples for Annexin V experiments. On the one hand, in the process of preparing tissue samples into single-cell suspension, the apoptosis rate caused by the experimental operation may be higher than that of the sample itself. Moreover, the experimental operation is unstable, and the actual apoptosis rate of the sample itself cannot be determined by the final experiment. On the other hand, tissue samples have multiple cell types, each with different autofluorescence, and the results cannot be analyzed.
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The FITC is 490nm/530nm and the 7AAD is 546nm/647nm (which can be excited with 488nm). Generally, it can be detected by FITC channel and PerCP channel.
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Untransfected cells with obvious apoptosis were stained with 7-AAD and APC, respectively, as single positive control 1 and single positive control 2. The samples transfected with green fluorescence were directly put on the machine as single-positive control 3.
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The cells in the control group can be induced apoptosis by the following two methods: ① Since different cells are affected by DMSO to different degrees, it is recommended to explore the action time or volume fraction of DMSO through pre-experiment. The action time can be explored according to the following volume fraction (10%) first, and the microscopic examination is conducted every 30 minutes. ② The cells were bathed in a water bath at 50~60℃ for 5~10min. When the cell morphology changes or a large number of floating, can be considered to produce apoptotic cell population.
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Because during the culture process, apoptotic cells do not stick firmly to the flask, and a large number of them float in the culture medium. For the accuracy of the results, it is usually recommended that the suspended cells be collected and tested together with the cells digested by trypsin when detecting apoptosis.
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We haven't tested it. Because single-celled diatoms have cell walls, it is recommended that customers do pre-experiments to explore the dyeing time and conditions.
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After the cells were suspended by PBS, anhydrous ethanol pre-cooled by -20°C was added drop by drop, and the cells were dispersed into single cells by shaking while adding. The fixed time must be at least 1 hour.
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In theory, placing it in a -20℃ refrigerator for 2-3 days after fixing has little impact on the results.
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If the sample does not contain fluorescence, the red, green and blue fluorescence cell cycle kit all can be used, and the red fluorescence kit (E-CK-A351) is more commonly used. If the sample has green fluorescence, use the cell cycle blue fluorescence kit (E-CK-A353) without adjustment compensation. If the sample has red fluorescence, it is recommended to use cell cycle blue fluorescence kit (E-CK-A353) and cell cycle green fluorescence kit (E-CK-A352). If the sample has red and green fluorescence, the cell cycle blue fluorescence kit (E-CK-A353) is recommended. Before selecting E-CK-A353, check whether the flow cytometer has a DAPI channel.
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The fluorescent dyes used are not the same, so the flow cytometry detection channel requirements are different from the applicable samples. The detection channel of red fluorescence is PI,PerCP and PE, the detection channel of green fluorescence is FITC, and the detection channel of blue fluorescence is DAPI.
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Heavy metal ions such as silver ions, copper ions, halogen ions and oxidizing organic compounds (nitro compounds, diazo compounds, carbonyl compounds and hydroxyl compounds) will quench the fluorescence of the fluorescent probe, thus affecting the accuracy of the experimental results.
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100 assays is calculated according to 96-well plate with 100μL working liquid per well, 24-well plate with 200μL working liquid per well, about 50 wells can be test.
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Matrix glue should be removed, not removed may interfere with calcein and cell binding; However, our calcein kit has not been verified by organoids, it is not clear what the final effect is, it is recommended to do a pre-experiment to see the actual effect.
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It's theoretically possible, but we haven't tested it, it is recommended to do a preliminary experiment to explore the details of the experiment.
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The cells of double positive are living cells in a poor state because the increased permeability of the cell membrane causes the nuclear dye to enter the cell. The cells of double negative are large granular cell fragments.
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The protein concentration was 1-4 mg/mL, and the caspase activity ranged from 2 U/ng prot to 430 U/ng prot.
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After the tissue sample is dry frozen, basically the cell morphology may have been destroyed and can not be used for flow detection. You can look at ELISA or metabolic indicators related to research, such as apoptosis of Bax, Caspase or oxidative stress of SOD, MDA and so on.
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① The microscope parameters are consistent; ② Dissolve the buffer and observe whether there are small red particles. If the buffer does not dissolve, centrifuge at 12000rpm and take the supernatant. (3) Apoptotic cells stick weakly to the flask and will float in the medium. If fluorescence microscopy is used, the results are relatively inaccurate, and flow cytometry is recommended for detection. Before collecting cells, take a photo at 100x magnification under normal white light (200x for small cells, such as lymphocytes) to check morphological changes.
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We do not recommend using tissue samples to prepare single-cell suspension for JC-1, because the operation of preparing single-cell suspension has a great impact on cells and will directly affect the experimental results of JC-1.
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It's theoretically possible, but we haven't tested it yet. We cannot determine the amount of JC-1 and the amount of extracted mitochondria, and may need to explore optimization.
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The monomers and polymers of JC-1 exist in both normal and apoptotic cells. In the process of cell apoptosis, JC-1 slowly dissociates from polymer to monomer, and the ratio of polymer to monomer changes. Therefore, when drawing the door, it is generally gathered in the third quadrant, and when drawing the door to distinguish between normal cells and apoptotic cells, it needs to be divided in the third quadrant.
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Generally, the absorbance value of CCK-8 is recommended to be about 1, preferably 0.8 to 1.2.
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It is recommended to use a transparent flat bottom, and the independent column is relatively suitable.
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Agreed. Because single cells in flow assay are more sensitive and fragile, the permeabilization operation should be mild. If customers do not have saponin, they can add 1%BSA or 3-5% FBS to PBS, and then add triton X-100 with a final concentration of 0.2%, and mix it as a permeabilization reagent.
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The addition of BSA can increase the osmotic pressure and ion concentration of PBS, making it more similar to the environment of intracellular fluid, thus reducing the effect of shear force on cells. In addition, BSA also has a certain buffer performance, which helps to maintain the stability of PBS, prevent the pH value of the solution from changing, and further protect the cells.
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The permeability of saponin is reversible, and the saponin permeability experiment is generally used, and the subsequent washing steps will continue to add saponin to maintain cell permeability.
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Theoretically, it can be done, but we have not used it. We suggest that you make sure whether this instrument can scan its absorption spectrum (230 nm~800nm) and record the data of A280 and A655 (1 cm optical path).
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150,000 is the molecular weight of the antibody, and the molecular weight of different subtypes of antibodies is different. 1 Dalton =1g/mol, the unit of molar extinction coefficient is (L/mol•cm). If the customer needs to use this formula to calculate the protein concentration, they also need to know some fixed parameters of their own protein.
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Fluorescence co-localization is the use of fluorescent probes and co-localization markers to link proteins to target sites, and our labeling kit allows fluorescein to bind to proteins to visualize the location of proteins. The binding site is the primary amino group of the protein for labeling, but whether fluorescence colocalization can be performed is related to the protein itself, or it needs to be determined by relevant pre-experiments.
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The labeling kit can effectively label proteins containing primary amino (-NH2) molecules, but the experimental application direction of the labeled proteins cannot be determined. It is recommended that customers do pre-experiments to verify the feasibility of the experiments.
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① The dewaxing solution after dilution needs to be reheated at 60℃, and low temperature affects the dewaxing effect; (2) It is recommended that the thickness of the paraffin section is 3~5μm, and the dewaxing time can be properly prolonged with the increase of thickness; ③ existing use; ④ The section should be completely immersed in the dewaxing solution; ⑤The melting point of paraffin from different sources is different (generally at 56~64°C), and this dewaxing solution is not suitable for paraffin sections with a melting point higher than 60 ° C.
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Both systems have the same resolution ratio, while one-step operation takes easier process, and two-step operation requires less reagent.
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It is not suitable. The emmision fluorescence of EGFP will interfere both FITC and PE channel which are the two channels for JC-1 detection.
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The method hasn't been verified.
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No. The detection must be done in one hour after staining. The permeabilization and mitochondrial membrane potential will be changed by fixation.
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JC-1 detects the change of mitochondrial membrane potential which happens during the early stage of apoptosis, TUNEL reflects the situation of DNA fragmentation during the late stage of apoptosis. A JC-1 positive but TUNEL negative result shows that the cells were in the early stage of apoptosis.
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The equilibrition process is to make a better working condition of TdT enzyme that benefit a better staining result.
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The best linearity of CCK-8 is between 0.2-2.5 (Optical Density).
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Yes it is. But Triton X-100 has more acurate effect, 0.2% Triton X-100 and 3% BSA in PBS could be used to protect cells.
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Normally, it is better to detect in 1-2 hours after staining. If the cells required further incubation, it can be kept in CO2 incubator with no more than 48 hours. When using microscope, it is recommanded to turn the illumination down and extend exposure time.
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It is better to start with the lowest illumination and lowest exposure time, then gradually increase the exposure time and fluorescence brightness. Over time exposure can easily cause dye bleaching and overexposure. It is better to keep the negative control without background fluorescence and make the positive control with well resolution. The exposure conditions for the same fluorescein in different groups need to be consistent.
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It is possible. But it have to be notice that the reagents must fully cover the sample and keep moist. It may require more reagent than the recommanded amount. The result should be observed by inverted microscope and cannot be sealed.
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Half dose reagent can be used with no more than 5*10^5 cells following the two-step operation.
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BSA is used for protecting cells from mechanical damage and losing during centrifuge and washing, especially for suspension cells.
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Yes, it is. The sample can be fixed with 2-4% paraformeldehyde for 30-60 minutes in room temperature, then kept with PBS in 4 ℃. The sample can be stored for no more than 3 days.