Cell scraper Vs. cell lifter
Both cell scraper
and cell lifter
are primarily used to collect cells. The blade of them is designed to greatly reduce damage to the cells and allow access to the growth surface. The blade is usually made of polymer material TPE, and the hilt is usually made of polymer material ABS. These materials are suitable for harvesting cells.
is mainly designed for use in cell culture
dishes for its wide beveled edge.
Cell scraper with angled, rotating blade for easy access to the growth surface of flasks and Petri dishes.
Features of cell scrape & cell lifter
a. For manual harvesting of cells.
b. Blade design makes smooth contact with the growth surface area, resulting in minimal damage to cells.
c. Gamma radiation sterilization.
d. Non-pyrogenic, non-endotoxic, non-cytotoxic.
e. Different lengths of knife handles meet the requirements for culturing different cell culture collections.
f. Individually wrapped.
g. Lab bottles available.
h. Different size for options: 18cm, 25cm, 40cm, mini cell scrapers, etc.
i. Used with well plates, such as cell scraper for 24 well plate, cell scraper for 12 well plate, etc.
Applications of cell scrape & cell lifter
In the pharmaceutical industry, cell culture has become an important component of antibody production. Cell culture technology has been used to produce antibodies related to rotavirus, polio, smallpox, hepatitis, rubella, and varicella. Cell-based antibodies against influenza have also been approved for use in the United States and many European countries. Here are a few examples of cell scrape & cell lifter applications.
Cell passage cultivation
The growth and proliferation of cells grown against the wall are hindered by the fact that the space is largely saturated after the culture flask or culture dish has grown to a monolayer spread. Cell passage cultivation
is performed to allow for continued cell expansion or growth. Also, passaging cultures serve to preserve cell
species. Besides, a variety of cell experiments are based on cell passage cultivation.
Suspended cells can be directly divided into bottles, while walled cells need to be digested and other treatments before they can be divided into bottles. Trypsin is generally used to digest the adherent cells into individual cells, and EDTA can also be added to improve digestion. EDTA is a chelator of divalent ions that inhibits Ca2+ and Mg2+ on the cell membrane. The commonly used cell digest solution generally contains 0.25% (w/v) trypsin and 0.03% (w/v) EDTA.
During the experiment, you should aspirate the cell culture supernatant, wash the cells with PBS, discard the washing solution, and re-add a small amount of trypsin-EDTA digestion solution. Depending on the size of the culture dish, 2 ml to 5 ml of digestion solution is sufficient to spread the entire bottom of the dish. The cells are then observed until they become round and detach from the bottle wall, which usually takes 3-5 minutes. To avoid clumping of cells, you should not tap the flask during the digestion process. For cells that are particularly difficult to digest, you can add trypsin EDTA digestion solution and then place the cells at 37°C to facilitate digestion. After the cells have been detached from the wall of the bottle, you should immediately add complete medium containing serum to abort the digestion.
Centrifuge the cells at 800-1000 rpm for 5 minutes. Then discard the discontinued medium, add a suitable new medium and gently mix the cells and transfer them to a new culture flask. The percentage of passaging depends on the type of cell. Trypsin could cause damage to the cell membrane of some cells. For this type of cells, you can gently scrape off them with a cellscraper
, add the appropriate amount of medium, gently blow to mix the cells, and then pass them through the culture.
Extraction of cellular proteins
A. Experiment preparation
a. Confirm that the ice machine is turned on and that there is enough crushed ice for use during operation.
b. Confirm that the ice machine is turned on and that there is enough crushed ice for use during operation.
c. Confirm that the enzyme marker is working properly, make an appointment and register before use.
d. Prepare 4 sets of 1.5 mL centrifuge tubes. They are used to collect lysed cell suspension, collect protein supernatant after centrifugation, determine the concentration of protein samples after 5-fold dilution, and determine the concentration of protein samples after 10-fold dilution.
e. Prepare cell lysis solution.
Before use, mix the cell lysis solution in the ratio of lysate 1mL: protease inhibitor (PMSF) 10μL. PMSF is effective within 30min, so pay attention to the ready-to-use preparation.
Calculate the amount of operating fluid that needs to be prepared in advance. The specific amount of operating fluid needs to be figured out on a case-by-case basis. Generally speaking, if the cells reach 70~80% confluence in a 25T culture flask, 250~350μL of working solution is needed for use. If you add too much cell lysis solution, the concentration of the final sample protein may be too low. If insufficient cell lysis solution is added, the cell lysis is not sufficient and the sample is sticky-like easily blocking the tip of the 200 μL gun. Cell lysis operating fluid is recommended to be pre-cooled at 4°C.
f. Prepare cell scrapers and cell lifters. At least 3 pieces of them for alternating when scraping cells.
g. Prepare three beakers of 100 mL or larger volume. These beakers need to be pre-washed and filled with ultrapure water. They are used for sequential washing of used cell scrapers. Both the rubber cell scraper and plastic cell scraper are available. Rubber policeman cell scraper is more recommended.
h. Prepare equilibrium salt solution.
i. Prepare a BCA protein concentration assay kit.
j. Prepare SDS loading buffer. It is stored at -20℃ and should be opened in advance before use.
B. Experimental procedure
a. Neat lab bench top
No matter what kind of experiment you are doing, it is essential to keep your operating table clean and tidy. When manipulating experiments like protein extraction or DNA/RNA extraction, it is recommended to operate in an ultra-clean table. In the absence of an ultra-clean table, we should ensure that the operating table is as neat as possible. Also, keeping the operating table neat is a good habit that needs to be developed for any experimental operation.
When confirmed the needed lab instruments for the experiment, the cells can be removed from the cell compartment.
b. PBS washing
After removing the cells, you should wash them with PBS at room temperature. Then all operations should be performed on an ice bath.
c. Cellular lysis
Add 250 to 350μL of lysis solution to each vial of cells. The lysate should be spread well and lysed in an ice bath for 10 min. You should repeat to spread the lysate consistently during this 10 min. The lysate spreading technique is the same as for trypsin digestion. While waiting for lysis, you should reconfirm the temperature of the centrifuge, thermostat, and water bath.
d. Cell scraping
After lysis is complete, you should scrape off the cells using a cell scraper. Each scraping action of the cells should be sticky with lysis solution, keeping one direction from top to bottom, and the action is like scraping and washing glass.
e. Transfer samples
Use a 200 μL pipette to transfer the scraped cell suspension to a 1.5 mL centrifuge tube (immediately after scraping a sample) and repeatedly blow 30-60 times to fully break up the cells. Try to avoid air bubbles during the blowing process. After the blowing is completed, the centrifuge tube with the sample should be inserted on ice.
f. Repeat step e. to complete sample collections
You should rinse the cell scraper and cell lifter used in step e. in three beakers in turn, shake cell scrapers gently and insert them on a test tube rack to dry. If cell lifters are not cleaned, this may lead to cross-contamination of the samples. If cells are not dried, resulting in sample dilution.
g. Further ice bath lysis
After samples are all scraped and transferred to centrifuge tubes, they are continued to be lysed in an ice bath for 5-10 min. During this period, the condition of the centrifuge, thermostat, and water bath should be reconfirmed.
After lysis is completed, centrifuge at 12,000g, 4°C, for 5 min.
i. Protein supernatant collection
Take the supernatant and use it for preparing protein concentration measurement and denaturing samples for WB. When taking the sample supernatant for WB, please record the sample volume and add the corresponding volume of SDS loading buffer subsequently according to the sample volume to prepare the cooking sample.
C. BCA method for protein concentration determination
a. The thermostat should be preheated to 37°C in advance.
b. Configure 5× and 10× protein quantification solution and stand aside on ice after preparation. Here it is recommended to use PBS for sample dilution, because the color of D-Hanks itself may affect the final result of protein concentration quantification.
5× dilution: 80 μLPBS + 20 μL supernatant.
10× dilution: 90 μLPBS + 10 μL supernatant.
c. Prepare the BCA protein quantification kit and calculate the total amount of operating fluid as needed. Prepare the operating fluid according to the instructions in the ratio BCA: Cu= 50: 1. Do not forget to mix the liquid completely. The mixed working solution is a clear, light blue color.
d. The 96-well plate is spiked with 20 μL per well, and at least three replicate wells are set up. Because the protein sample is very viscous and the spiked volume is small, the tip of the gun can be extended into the well and spiked against the bottom of the well to complete the spiked sample by using the liquid tension.
e. After the sample addition is completed, each sample well is supplemented with 200 μL of the operating fluid. The action of adding operating fluid should be quick and clean. Quick and clean addition of operating fluid facilitates adequate mixing.
f. Incubate for 30 min at 37°C. After the reaction is completed, the assay should be completed within 3~5 min.
g. The absorbance is measured at the specified wavelength by an enzyme marker.
h. The protein concentration should be calculated according to the equation of the standard curve that has been previously prepared. See the kit instructions for the method of plotting the standard curve.
D. Denaturation of protein samples for WB
a. Preheat the water bath to boiling point in advance to avoid dry boiling and pay attention to safety.
b. Use 5× protein loading buffer, which is stored at -20°C, dark blue, and dissolve it before use.
c. In each group of protein supernatant samples, you should make up the corresponding volume of loading buffer (protein supernatant: buffer = 4:1) separately and blow well.
d. Each sample tube should be clamped on the explosion-proof clip, inserted into the sponge float, denatured by boiling water for 5 min, cooled by cold water for 1 min, and repeated three times to ensure that the protein samples were thoroughly denatured.
e. The denatured protein should be uniformly blue, clear, and transparent. The sample should be slightly centrifuged, stored at -20°C, and set aside.
f. Till this step, this experiment is completed.
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