Looking for Cell Counting Kit-8 protocol?

The Cell Counting Kit-8 is a colorimetric assay kit used to measure cell proliferation and cytotoxicity. It is a ready-to-use solution that can be added directly to the cell media for fast, high-throughput screening without a solubilization process obtaining highly reproducible and accurate results.

Cell Counting Kit-8 can be utilized to do cell number counting, cell prolifiration or cytotoxicity assay based on your requirments.  So it make a tiny protocol deferences when you apply different assay, while the majority of  operation protocol are the same. However, Some equipment and materials should be prepared before assay: 1). Plate reader (450 nm filter), 2). 96-well plate, 3). 10 µl, 100-200 µl and multi-channel pipettes.

 



More detailed manipulation procedures for Cell number counting and Cell proliferation and cytotoxicity sssays from Abbkine CCK-8 Kit are listed below for your references,

Cell Number Determination Protocol

1) Inoculate cell suspension (100 µl/well) in a 96-well plate. Also prepare wells that contain known numbers of viable cells (to create a calibration curve in step 5). Pre-incubate the plate in a humidified incubator (e.g., at 37 °C, 5% CO2).

2) Thaw the CCK-8 on the bench top or in a water bath at 37 °C if it is frozen. Note: It takes about 30 minutes on the bench top at 25 °C or 5 minutes in a water bath at 37 °C.

3) Add 10 µl of the CCK-8 solution to each well of the plate. Note: Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.

4) Incubate the plate for 1-4 hours in the incubator.

5) Measure the absorbance at 450 nm using a microplate reader. Prepare a calibration curve using the data obtained from the wells that contain known numbers of viable cells.

Note: To measure the absorbance later, add 10 µl of 1% w/v SDS to each well, cover the plate and store it with protection from light at room temperature. No absorbance change should be observed for 48 hours.

Cell Proliferation and Cytotoxicity Assay Protocol

1) Dispense 100 µl of cell suspension (5000 cells/ well) in a 96-well plate.

2) Pre-incubate the plate for 24 hours in a humidified incubator (e.g., at 37 °C, 5% CO2).

3) Add 10 µl of various concentrations of toxicant into the culture media in the plate.

4) Incubate the plate for an appropriate length of time (e.g., 6, 12, 24 or 48 hours) in the incubator.

5) Thaw the CCK-8 on the bench top or in a water bath at 37 °C if it is frozen. Note: It takes about 30 minutes on the bench top at 25 °C or 5 minutes in the water bath at 37 °C.

6) Add 10 µl of CCK-8 solution to each well of the plate. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.

7) Incubate the plate for 1-4 hours in the incubator. Measure the absorbance at 450 nm using a microplate reader.

Note: To measure the absorbance later, add 10 µl of 1% w/v SDS to each well, cover the plate and store it with protection from light at room temperature. No absorbance change should be observed for 48 hours.

Be attention to conditions or chemicals that affect dehydrogenase activity in viable cells may cause discrepancy between the actual viable cell number and the cell number determined using the Abbkine CCK-8 assay.

Anti-Nano-Tag9 Monoclonal Antibody(11T3) Review

The Nano-tag is a new streptavidin-binding peptide for both the purification and the detection of Nano-tagged proteins. This peptide possesses nanomolar-affinity for streptavidin and therefore is termed Nano-tag. The nano-tags have two types, Nano-tag15 (MDVEAWLGARVPLVET) and Nano-tag9 (MDVEAWLGAR), which bind to streptavidin with dissociation constants of 4 nM and 17 nM, respectively. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the Nano-tag(9) is a 9-mer peptide with a dissociation constant of 17 nM.

Anti-Nano-Tag9 Monoclonal Antibody(11T3) was affinity-purified from mouse ascites by affinity-chromatography using specific immunogen. This antibody has been tested with WB. And Abbkine suggested starting dilutions are as follows: WB 1:2000-5000.

Well-characterized antibodies for epitope tags consisting of short sequences are widely used in the study of protein expression in various systems. We find the quality suitable for our experiments. This product is what I want.

 

Choose right VSV-G Tag Antibody?

Epitope tags include VSV-G tag, Myc-tag, HA-tag and NE-tag. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification.

If you’re working in the life sciences with VSV-G tag in your vector or fusion proteins, chances are you’re going to be using right VSV-G antibodies in your research. Here we've set out some top tips to get you started in making a good and suitable VSV-G tag antibody buying decision.

Is this VSV-G antibody suitable for your application?

It's “impossible” to predict which antibodies will work and which will not.  Just because an antibody has been successfully used in one application does not mean it will work for all applications. That means excellent WB and IHC results do not tell you whether the antibody is the right choice for a so-called ChIP-seq experiment, in which chromatin immunoprecipitation is followed by second-generation sequencing.

You must choose application-specific validation of VSV-G antibody you use. However, due to the price and performance, researchers prefer the antibody with more validated applications, especially these kinds of epitope antibodies.

Decision on monoclonal or polyclonal VSV-G tag antibody

Our advice is you should go with the most validated antibody, but if you are lucky enough to have two well validated antibodies to chose between, then polyclonal antibodies tend to be cheaper because they are raised against multiple epitopes on your protein and are therefore less specific. Monoclonal are more expensive and take longer to produce, however they are specific to a single epitope. Monoclonal antibodies are most useful if you are trying to identify specific isoforms or region of a full-length protein, especially in tag fusion detection.

[caption id="attachment_14" align="aligncenter" width="384"] Immunofluorescence staining of VSV-G fusion protein with VSV-G monoclonal antibody (Clone #14D2) in 293 cells with red and counterstained with DAPI.[/caption]

A manufacturer may license their antibody to other companies, so do not buy three antibodies to test without making sure they are actually different. With common monoclonals that are supplied by many companies you might also consider shopping around for the best supplier of a clone.

Finally, remember you should feel free to contact the company supplier for advice any time. The suppliers really should be helpful, many pride themselves on their customer service, and if they are not you can remember that for next time you buy an antibody.

If you are looking for a detection tool to quantify or localize your VSV-G fusion proteins, we highly recommend you to try our featured monoclonal VSV-G Antibody risk free. This antibody with clone number 14D2 can specifically recognizes native and denatured forms of VSV-G fusion proteins, with which can meet your any requirements for Western Blot, Immunofluorescence and Immunoprecipitation assays.

Preparing Cell Counting Kit-8 Solution ?

Cell Counting Kit-8 (CCK-8) solution in kit assign very convenient assays by utilizing highlywater-relaxed tetrazolium sailor, WST-8 to produce a water-dissolvable formazan Dyeupon decrease in the person of an electron porter, as shown in below.  WST-8 is educed by dehydrogenases in cells to give a yellowcolored roduct (formazan), which is soluble in the membrane civilization medium. The amount of the formazan dye generated by the nimbleness of dehydrogenases in cells is soon relative to the number of ignited cells.

"The CCK-8 Kit has been optimized for use." Said Brooke Kilyanek, Technical Manager of Abbkine Scientici. "If you want to buy WST-8 and 1-methoxy-PMS, and then mix the two regents  together to Cell counting kit-8 solution for cell proliferation assays yourselves. This is also certainly an option, but the best protocol to use is ultimately up to the researcher. "

Cell Counting Kit-8 is a one-deadman solution, and no mixing of components is required, which is totally differently from other commercial player in the market, such as WST-8 Kit. WST-8 Kit is normal made from two solution components. One component is an electron carrier for NADH-tetrazolium, which is 1-methoxy-PMS in most cases. And another component is WST-8 solution with proper reaction buffer.  While Cell Counting Kit-8 featured only one optimized solution component, which is nonradioactive, allot sensitive colorimetric assays for the purpose of the reckon of viable cells proliferation and cytotoxicity risk.

Since the CCK-8 solution is very stable and it has weak cytotoxicity, a longer incubation, such as 24 to 48 hours, is possible. We highly suggest you purchase bulk size of Cell Counting Kit-8 if you're willing to prepare CCK-8 solutions yourself. Since this will not only saving your time and cost, but also ensuring you reliability of cell research. Believe me, high quanlity branded CCK-8 allows you archieve colorimetric assays for the determination of the number of viable cells in the proliferation and cytotoxicity assays with high performance and high throughput.

How to Choose right Cell Counting Kit-8 product

Choosing a cell viability or cytotoxicity assay from among the many different options available can be a challenging task. Picking the best assay format to suit particular needs requires an understanding of what each assay is measuring as an endpoint, of how the measurement correlates with cell viability, and of what the limitations of the assay chemistries are.

Cell Counting Kit-8 allows sensitive colorimetric assays for the determination of the number of viable cells in the proliferation and cytotoxicity assays. CCK-8 highly water-soluble tetrazolium salt, WST-8, is reduced by dehydrogenase activities in cells to give a yellow-color formazan dye, which is soluble in the tissue culture media. Cell Counting Kit-8 is a one-bottle solution; no premixing of components is required. Cell Counting Kit-8, being nonradioactive, allow sensitive colorimetric assays for the determination of the number of viable cells n cell proliferation and cytotoxicity assays.

PurKine™ Anti-Flag Resin 4FF Released

FLAG-tag,  is a polypeptide tag that can be added to a protein using recombinant DNA technology, having the sequence DYKDDDDK. It has been used for studying proteins in living cells and for protein purification by affinity chromatography. It has been used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. Because of the small size of the FLAG peptide tag, it is not likely to obscure other epitopes, domains, or alter function, secretion, or transport of the fusion protein. The peptide sequence of the FLAG-tag from the N-terminus to the C-terminus is: DYKDDDDK (1012 Da).

PurKine™ Anti-Flag Resin 4FF has been designed for affinity purification of Flag fusion proteins.The Resin consists of 90μm beads of cross-linked 4% agarose, to which mouse monoclonal antibody against DYKDDDDK tag has been coupled. Tests confirm that performance equals or exceeds popular Anti-Flag resins from other suppliers, and no decrease in performance occurs after at least five repeated uses. In addition, its high flow properties make it excellent for scaling-up.

More than 1mg DYKDDDDK-tagged protein can be captured by per mL of PurKine™ Anti-Flag Resin 4FF. The maximum tolerance pressure of the resin is 0.3MPa, 3bar, which is excellent for industry production. In addition,  Anti-Flag Tag Mouse Monoclonal Antibody (1B10) is a good option to detect the Flag fusion protein that is affinity purified.

Anti-AmCyan Monoclonal Antibody (8T2) Review

AmCyan is one of the existing violet fluorochromes for use in flow cytometers equipped with a violet (405 nm) laser. It is also widely used as a label in fluorescent spectroscopy. AmCyan was originally derived from the fluorescent protein amFP486. AmFP486 was cloned from the sea anemone Anemonia majano (GenBank accession number AF168421), and belongs to the family of fluorescent proteins (FPs) isolated from coral reef organisms; green fluorescent protein (GFP), derived from the jellyfish Aequorea victoria, is its most famous representative.AmCyan has been
adapted from the corresponding full length cDNA for higher solubility, brighter emission, and rapid chromophore maturation. AmCyan1 has been human-codon optimized for enhanced translation in mammalian cells.

Anti-AmCyan Monoclonal Antibody (8T2) was affinity-purified from mouse ascites by affinity-chromatography using specific immunogen. This antibody has been tested with WB. And Abbkine suggested starting dilutions are as follows: WB 1:5000.

AmCyan is a very good blue fluorescent reporter protein, and useful in two-color analyses with DsRed2, DsRed-Express, or AsRed2, and in three-color analyses with ZsYellow and HcRed. I've tried Anti-AmCyan Monoclonal Antibody (8T2) with excellent results. I am a loyal fan of Abbkine！