Carp Epithelial Tumor Cell Line (EPC)

Product Name:Carp Epithelial Tumor Cell Line (EPC)

Product Category: Other Cell Lines

Growth characteristics: Wall attached growth

Cultivation system: MEM+10% FBS, cultured at 28 ° or 21 ° C

Passage method: 1:2 passage

Cell morphology: Epithelial like

Specification: 1 × 10 ^ 6 cells/T25 culture bottle

Item number: CS-C2008

Methods for cryopreservation of cells:
Cryopreservation method one: Place the freezing tube in a liquid nitrogen vapor phase tank at 4 ℃ for 30-60 minutes (-20 ℃ for 30 minutes *) -80 ℃ for 16-18 hours (or overnight) for long-term storage.
Freezing preservation method 2: Place the freezing tube in a programmable cooling machine that has been programmed to cool down 1-3 ℃ to below -80 ℃ per minute, and then store it in a liquid nitrogen tank vapor phase for long-term storage. *-At 20 ℃, it should not exceed 1 hour to prevent excessive ice crystals from causing massive cell death. This step can also be skipped and placed directly in a -80 ℃ freezer, but the survival rate is slightly reduced.

Epidermal tissue is formed from the embryonic ectoderm and has anti friction and anti damage properties. The company's technology provides high-quality total RNA, PCR prepared cDNA strands, proteins, and their sub components from fresh or frozen rat fetal epidermal tissues. All products undergo strict QA/QC processes before shipment to ensure their quality. These products can be directly used for gene cloning, expression profiling analysis, and various molecular biology experiments.
Total RNA preparation:Use the Trizol reagent of Qiagen RNeasy Kit to isolate RNA, and then purify it using Qiagen RNA Clean Kit. The total RNA samples provided by the company are accompanied by RNA sample total concentration, total content, electrophoresis photos, OD value determination results, etc.

CDNA preparation:The purified total RNA was used to synthesize cDNA chains, and reverse transcription was performed using a 50ul total reaction system, which included MMLV reverse transcriptase, random primers, and 10mg total RNA. Terminate the RT reaction after 10 minutes at 68 ℃. Using 1x RT Buffer to transport cDNA, 1 ul of cDNA is sufficient for one PCR reaction. All cDNA products undergo strict QA/QC analysis before shipment to ensure product quality.

Protein preparation:Utilizing Improved Models RIPA Buffer (50mMTris, pH7.2, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% NP-40 , 1mM Na3VO4, 5mM NaF, 400uM PMSF, 1ug/ml of Pepstatin A, 5ug/ml of Aprotinin, 5ug/ml of Leupeptin) Prepare rat fetal epidermal tissue lysate, centrifuge to remove tissue fragments, measure protein concentration using Bio Rad protein detection kit, dilute tissue protein, add 5% β - mercaptoethanol and PMSF to non reducing protein buffer (50 mM Tris HCl, pH 6.8, 5% Glycerol, 1% SDS, 0.002% Bromphenol Blue), and store at -80 degrees Celsius.

Potential applications: <1> PCR cloning of known genes; <2> MRNA alternative splicing; <3> Gene cloning and target sequencing; <4>Western and Northern Blot； <5> Protein detection and proteomics; <6> Chip research and expression profiling.

Cell processing:

1) Resuscitate cells: Quickly place a cryovial containing 1mL of cell suspension into a 37 ℃ water bath (with the water level lower than the lid of the cryovial) and shake to thaw. Transfer the cryovial into a 15ml centrifuge tube containing 4mL of culture medium prepared in advance and mix well. Centrifuge at 1000RPM for 4 minutes, discard the supernatant, add 1mL of culture medium and blow evenly. Then transfer all cell suspensions into a culture bottle containing 5ml of culture medium and culture overnight. The next day, change the solution and check the cell density.
2) Cell passage: If the cell density reaches 80% -90%, passage culture can be carried out.
For suspended cells, the following methods can be used for passaging:
Method 1: Collect cells at 1000 RPM, centrifuge at room temperature for 5 minutes, discard the supernatant, add 1-2 mL of culture medium and blow evenly. Divide the cell suspension into new dishes or bottles containing 8 mL of culture medium in a ratio of 1:2 to 1:5.
Method 2: You can choose the method of half changing the medium. After discarding half of the medium, suspend the remaining cells and divide the cell suspension into new dishes or bottles containing 8ml of medium in a ratio of 1:2 to 1:3.
3) Cell cryopreservation: When the cells are in good growth condition, cell cryopreservation can be performed. When freezing suspended cells, the cells should be collected at 1000 RPM and centrifuged at room temperature for 5 minutes. A small amount of supernatant should be stored (to prevent cells from being sucked away), and some fresh culture medium should be added. After blowing evenly, it should be added to the freezing tube. 10% DMSO should be added to the freezing tube and shaken well before freezing. Frozen culture medium
When freezing cells, the recommended freezing culture medium must be used. The frozen culture medium should contain cryoprotectants such as DMSO or glycerol. Specially prepared frozen culture medium can also be used.
1) Cell cryopreservation medium is a ready to use type of cryopreservation medium for mammalian cells, which contains optimized ratios of fetal bovine serum and bovine serum to improve cell viability and cell recovery after thawing.
2) Cryopreservation medium is a chemically determined, protein free, sterile cryopreservation medium containing 10% DMSO, suitable for cryopreservation of various stem cells and primary cells (excluding melanocytes).
Operation process:
1.1 Main reagents and instruments: inverted phase contrast microscope (Olympus IMT-413, Japan), CO2 incubator (Yamato IT-61, Japan).
1.2 Recovery, culture, passage, and cryopreservation of hek-293 cells:
1.2.1 Cell Resuscitation Culture: Remove the cryovial from the liquid nitrogen tank, immediately immerse it in a 37 ℃ water bath, and shake continuously to rapidly melt it. Remove the dissolved cell cryopreservation tube, disinfect it with alcohol, open it, aspirate the cryopreservation solution containing cells, add it to a centrifuge tube pre filled with culture medium (preheated at 37 ℃, 8-10 times the volume), gently blow and mix, then remove the supernatant at 1000rpm for 5 minutes, add an appropriate amount of culture medium, blow and beat into a cell suspension, inoculate it into a 25cm2 culture bottle at a density of 1 × 105/ml, and culture it at 37 ℃, 5% CO2, and saturated humidity.
1.2.2 Cell Passage: The hek-293 cells cultured in primary culture should be changed once every 48 hours. When the cells have grown to 60% to 70% fusion, they should be digested with 0.25% for 1-2 minutes. The culture bottle should be gently tapped with the palm of the hand to remove the cell wall, suspend, and disperse the cells. To further disperse the cells, a blow tube can be used to gently blow several times to obtain a cell suspension. Then, double the amount of culture medium should be added, gently mixed, and the mixture should be divided into pipettes for cell propagation and expansion.
1.3 Observation of Cell Morphology: Observe and record the growth and morphological characteristics of cells under an inverted microscope.
1.4 Counting of Cells Conventional digestion of cells, wait for the cells to become round, detach and float, add a small amount of culture medium for centrifugation, then resuspend with PBS and blow to make a cell suspension. Add a small amount of cell suspension on one side of the cover glass of the cell counting plate, observe the number of large square cells at the four corners of the counting plate with a 10 × objective lens, and calculate the number of cells according to the formula. Cell count/ml original solution=(sum of square grid cell counts/4) × 104.
The adhesion rate index of 1.5 cells during the growth phase was digested into a single-cell suspension at 0.25%, counted, and inoculated into 12 25cm2 culture bottles. Three bottles of cells were randomly taken every two hours, and the culture medium and non adherent cells were removed. Pancreatic digestive enzyme was added to digest and count adherent cells, and the average value was taken. The adhesion rate was calculated according to the formula: adhesion rate (%)=(number of adherent cells/number of inoculated cells) × 100%.
1.6 Growth Curve: Cells in the exponential growth phase were digested and prepared into a single-cell suspension. The suspension was seeded into a 24 well plate at a rate of 1 × 104/well, with 1ml of culture medium added to each well. Randomly select 3 wells every day, count the number of cells in each well, and calculate the average value. Count continuously for 10 days and plot the cell growth curve.

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Carp Epithelial Tumor Cell Line (EPC) Human lipase A2 ELISA kit ≥ 4190% as determined by SDS-PAGE 2,5-diphenylenediamine

Human glucose mutase ELISA kit ≥ 41.91% as determined by SDS-PAGE 4-chloro-6-yl-5-fluoropyrimidine

Human inositol 3-kinase ELISA kit ≥ 4192% as determined by SDS-PAGE L - (+) - almond ester

Humanized Adenosine Activated Protein Kinase ELISA Kit ≥ 4194% as determined by SDS-PAGE N, N '- (4,4' - phenylene) bismaleimide

Humanized extracellular signal regulated kinase ELISA kit ≥ 4195% as determined by SDS-PAGE ortho trifluorobenzene

Humanized nuclear factor kappa B inhibitor protein alpha ELISA kit ≥ 4196% as determined by SDS-PAGE with second-generation amino zinc

Humanized protein kinase C ELISA kit ≥ 4197% as determined by SDS-PAGE 2,4,5-trimethylthiazole

Human Fructokinase ELISA Kit ≥ 4198% as determined by SDS-PAGE 6-Hydroxy-2-Naphthalenesulfonate Sodium

Human Glycerol Mutase 2 ELISA Kit ≥ 4199% as determined by SDS-PAGE 4-fluoro-3-ylphenylaldehyde

Human lymphocyte factor ELISA kit ≥ 4200% as determined by SDS-PAGE 2,3-dipyridine

Human lymphocyte chemokine ELISA kit ≥ 4201% as determined by SDS-PAGE based 3-ester

Human lymphocyte antigen 6 complex locus A ELISA kit ≥ 4202% as determined by SDS-PAGE L-valinate salt

Human lymphocyte function associated antigen 3 ELISA kit ≥ 4203% as determined by SDS-PAGE 3,4,5-trifluorobenzonitrile

Human lymphocyte function associated antigen 2 ELISA kit ≥ 4204% as determined by SDS-PAGE 4-ylphenylboron

Experimental report:
1、 Separation and cultivation:
1. Under sterile conditions, extract atrial tissue from 1-3 day old SD rats, then wash the tissue block twice with PBS, and finally make it approximately 1mm3 in size;
2. Add 4 mL of enzyme digestion solution (0.1% 0.1% type I collagenase) to the tissue block, suspend for 10 seconds, digest at 37 ℃ for 10 minutes, then use a dropper to prepare a single-cell suspension, naturally precipitate and collect the supernatant, terminate digestion with 10% FBS medium, and place at 4 ℃;
3. Add 3-4mL of enzyme digestion solution to the remaining tissue, suspend for 10 seconds, digest at 37 ℃ for 10 minutes, collect the supernatant according to the above method, terminate digestion, and place at 4 ℃. Repeat this step 2-3 times until the tissue is digested;
4. Filter the cell digestion solution through a 200 mesh stainless steel sieve, centrifuge at 1200r/min for 10 minutes, discard the supernatant, and suspend the precipitated cells in DMEM/F12 medium containing 10% FBS. Inoculate the cells into a 25cm2 culture bottle and incubate in a 37 ℃, 5% CO2 incubator;
5. After 1 hour of differential adhesion, aspirate the culture medium and inoculate it into a 6-well plate as needed for further cultivation;
2、 Immunofluorescence identification:
1. When the atrial myocytes grow to 80% fusion, discard the culture medium and wash the cells twice with warm PBS for 10 minutes each time. Then fix the cells with 4% paraformaldehyde at room temperature for 15 minutes;
2. Wash the cells twice with PBS for 10 minutes each time, and then permeate the membrane with 0.1% Triton X-100 at 4 ℃ for 15 minutes;
3. Wash the cells twice with PBS for 10 minutes each time, and then block the cells with 4% BSA at room temperature for 30 minutes;
4. Dilute the alpha actin primary antibody in a ratio of 1:100, and then incubate the cells overnight at 4 ℃ in a refrigerator;
5. Wash the cells with PBS three times, each time for 10 minutes. Dilute the secondary antibody against alpha actin in a ratio of 1:150 and place it at 37 ℃ for 1 hour;
6. Wash with PBS three times for 10 minutes each time, then observe the image under an inverted fluorescence microscope and take photos.