Example experimental protocols
Note: adapt text shown in red to the actual experimental conditions.
Version 1: For GeneA, search Sigma’s website for shRNA sequences with validated knockdown efficiency. After synthesis, add restriction sites at both ends and synthesize a double-stranded DNA oligonucleotide to form an shRNA expression cassette with the required cohesive ends. Use EcorI and BamHI restriction enzymes to digest pGP vector to linearize it and generate asymmetric cohesive ends. Insert the shRNA cassette into pGP, transform competent E. coli, and identify positive clones by PCR and sequencing to obtain a GeneA-targeting shRNA plasmid.
Version 2: Retrieve the human GeneA nucleotide sequence from GenBank, design GeneA shRNA sequences using the Ambion database software, and use BLAST to assess homology. Select and label three relatively specific candidates and use a replaceable stem–loop structure as a negative control. Take pGP 15 μL of shRNA vector in a microcentrifuge tube; digest with 1.5 μL BamHI, 1.5 μL EcoRII and 5 μL 10× buffer for at least 4 hours. Recover the product and add T4 DNA ligase to join the GeneA shRNA fragment to the target vector; transform DH5α competent cells, extract plasmids with a miniprep kit, and identify positive clones by PCR and DNA sequencing.
Retrieve the target gene CDS from the PubMed Nucleotide database and analyze restriction sites in DNAMAN. Choose two enzymes present in the vector’s multiple cloning site but absent from the CDS: EcoRI and SacII. Design primers at the start and end of the GeneA coding sequence and add the corresponding restriction sites to their 5′ ends. Amplify the gene from cDNA, recover the fragment from the gel, digest it at both ends and purify it. Use the same restriction sites to digest the pCDH vector for linearization; gel-purify the vector, ligate it with the fragment and transform competent DH5α cells. Identify positive clones by PCR and sequence them.
Version 1: Design and synthesize two complementary oligonucleotide primers containing the desired mutation. Use PrimeSTAR® GXL DNA polymerase to synthesize the full mutant plasmid. Add DpnI and incubate at 37°C for 1 hour to digest the methylated original template while retaining the unmethylated mutant plasmid. Transform competent E. coli; gaps in the PCR-derived mutant plasmid are repaired in the cells, and the resulting colonies contain the mutant plasmid.
Version 2: Use the Hieff Mut™ Site-Directed Mutagenesis Kit to construct the GeneA point-mutation vector. Design primers containing the mutation and amplify the plasmid by inverse PCR. Mix the product with DpnI and digest at 37°C for 1–2 hours to remove the methylated template. Prepare the single-site recombination reaction with Exnase and incubate at 37°C for 30 minutes, then immediately cool on ice for 5 minutes. Add 20 μL of the cooled reaction to 200 μL competent cells for transformation; plate 100 μL on suitable antibiotic medium and identify positive clones by PCR and sequencing.
Find the GeneA CDS in NCBI and identify an exonic target; the original template specifies entering a 23–250 bp exon sequence into design software. Synthesize complementary oligonucleotides according to the sgRNA design, anneal by gradual cooling, ligate into the Cas9 plasmid, transform competent DH5α E. coli and sequence positive clones. The original text subsequently calls the expression element “shRNA”; this conflicts with its sgRNA description and requires verification. For the mismatch-nuclease activity assay, PCR-amplify the target, denature and reanneal to form mismatched heteroduplexes, digest with CEL1 or T7E1, and compare cleaved and uncleaved electrophoresis bands.
Use Clontech Lenti-XTM HTX packaging system, described in the original as fourth-generation. Approximately 24 hours before transfection, seed 4–5 × 106 Lenti-X 293T cells per 100 mm dish in 10 mL medium and culture overnight. According to the instructions, mix the DNA transfection and polymer systems separately, combine them and incubate for 10 minutes at room temperature, then add dropwise to 293T cells and mix gently in a cross pattern. Replace medium after 4 hours, continue culture for 48 hours, collect viral supernatant and centrifuge at 500×g for 10 minutes to remove debris; store at −80°C. Assess titer with Lenti-X GoStix™ or by serial dilution and 293T cell infection efficiency.
The original heading groups adenovirus and adeno-associated virus, while the following example describes adenovirus; their protocols should not be assumed interchangeable. Digest 5 μg recombinant plasmid with PacI and gel-purify the large fragment. Use TurboFect™ in vitro transfection reagent to transfect 293T cells at 80–90% confluence with 2 μg per well in a six-well plate. After 24 hours, observe green fluorescence to track adenoviral packaging and propagation. At 6–8 days, localized clusters of green fluorescence appear; at 10–12 days, cells round and detach with visible plaques. When about 50% have detached, collect all medium and cells, freeze and thaw the suspension between −80°C and 37°C with vortexing twice, then centrifuge at 5,000×g for 5 minutes. The supernatant is the first-generation viral suspension.
Infect 293A cells at approximately 90% confluence with the first-generation suspension. After 2–3 days, cells become rounded and detach; when about 50% have detached, collect cells and recover supernatant as above to obtain the second-generation suspension. Infect 293T cells with the second-generation suspension and repeat infection, freeze–thaw and collection to expand virus and increase titer. Determine the collected high-titer suspension using a GFP-based titer assay.
Cell pretreatment (Example: using gene knockdown to study its effect on cell proliferation): seed cells in six-well plates at 50,000 cells per well. Experimental groups: ① blank control, ② negative control, ③ experimental group. 37°C, 5% CO2 After overnight culture, replace the medium with OPTI-MEM and add the amount of virus calculated from the MOI. Mix gently and continue culture for four days, observing green fluorescence by fluorescence microscopy to assess expression.
1) MTT assay
Dissociate cells to prepare a single-cell suspension; seed at 2,000 cells/well in a 96-well plate with five replicate wells per group and measure for 5 days. Before measurement, add 20 μL of 5 mg/mL MTT prepared in PBS at pH 7.4 to each well and incubate at 37°C for 4 hours. Carefully remove the supernatant, add 150 μL DMSO per well and shake for 10 minutes to dissolve the crystals. Read absorbance at 490 nm using a microplate reader.
2) CCK-8 assay
Using a Cell Counting Kit (CCK-8/WST-8), dissociate cells to prepare a single-cell suspension and seed at 2,000 cells/well in a 96-well plate with five replicate wells per group and measure for 5 days. Before measurement, add 10 μL CCK-8 reagent per well and incubate for 2 hours at 37°C in the dark. Transfer the incubated medium to a microplate and measure absorbance at 450 nm using a BIO-RAD microplate reader.
3) BrdU assay
Cells at 1.5×105 cells/mL were seeded in six-well plates containing coverslips, with three replicate wells per group. After one day, synchronize for three days in medium containing 0.4% FBS so most cells enter G0. Add BrdU (stock 1.0 mg/mL; final concentration 0.03 μg/mL) and incubate at 37°C for 40 minutes. Remove the supernatant, gently wash coverslips two or three times with PBS, fix in methanol for 10 minutes and air-dry. Inactivate endogenous peroxidase in 0.3% H2O2–methanol for 30 minutes; block with 5% normal rabbit serum, denature nucleic acids in formamide at 100°C for 5 minutes, cool on ice and wash with PBS. Add mouse anti-BrdU primary antibody at 1:50; use PBS or serum for the negative control. Detect by the ABC method, counterstain with hematoxylin or eosin, randomly count total and BrdU-positive cells in ten high-power microscopic fields, and calculate the labeling index.
1) Plate colony formation (adherent cells)
Cells at 500 cells per well were seeded in six-well plates, mixed in a cross pattern and cultured with three replicate wells per group at 37°C in 5% CO2. Examine colony size by microscopy every two days and replace medium every three days. When most colonies contain more than 50 cells, photograph them by fluorescence microscopy. Remove medium, wash carefully once with PBS, add 0.1 mL of 10% methanol per well for 30 seconds, remove the methanol and add 0.1 mL crystal violet per well for 20 minutes at room temperature. Remove stain gently, wash with distilled water, invert the plate onto absorbent paper and air-dry or dry at 37°C.
2) Soft agar colony formation (suspension cells)
Dissociate cells into a single-cell suspension and adjust the density to 1,000 cells/mL. To prepare the bottom agar, mix fully melted 5% agar with cell suspension prewarmed to approximately 37°C at a 1:9 ratio at 40°C; add 2 mL of the resulting 0.5% agar medium per 60 mm dish and allow it to solidify at room temperature. For the upper layer, place 1.5 mL cell suspensions at different densities (50, 100 or 200 cells per dish) in a small beaker and mix with an equal volume of 5% agar at 40°C. The source calls this 0.25% semisolid agar, which does not match the stated equal-volume mixture and requires verification. Add it immediately over the bottom layer, allow it to solidify at room temperature and culture at 37°C in 5% CO2 for 2–3 weeks before observing colony formation.
Seed cells in 6 cm dishes at the source-stated 30% density. After 48 hours, dissociate with trypsin and collect; centrifuge at 12,000 rpm for 5 minutes and discard supernatant. Wash twice with cold PBS, add cold 70% ethanol and fix overnight at −20°C. Centrifuge at 1,500 rpm for 5 minutes, discard supernatant and wash once with 3 mL PBS. Add 250 μL propidium iodide (100 μg/mL) and 250 μL RNase A (100 μg/mL), then incubate for 30 minutes at 4°C in the dark. Filter through 200-mesh nylon before flow cytometry to reduce clumping. Analyze 20,000–30,000 cells on a Beckman flow cytometer and analyze the cell cycle with FlowJo. Perform three independent replicates per group.
1) Annexin V / 7-AAD flow cytometry
Use the KGA1026 kit. Dissociate cells with EDTA-free trypsin; stop digestion, centrifuge at 1,500 rpm for 3 minutes, remove supernatant, wash once with PBS and resuspend for counting. Centrifuge a suspension of 1 × 10^5 cells, discard supernatant and resuspend in 500 μL binding buffer. Add 5 μL Annexin V-APC and mix, then add 5 μL 7-AAD and mix. Incubate for 5–15 minutes at room temperature in the dark, place on ice and analyze by flow cytometry within 1 hour. Use the FITC channel to select GFP-positive cells for Annexin V-APC/7-AAD analysis. The original text lists Annexin V-APC excitation at 633 nm and maximum emission at 660 nm, and 7-AAD excitation at 546 nm and emission at 647 nm.
2) DAPI staining
Rinse cultured unfixed cell monolayers or frozen fresh-tissue sections in PBS for 5 minutes. Prepare DAPI stock by dissolving 0.5 mg in 5 mL PBS, aliquot and store cold; dilute in PBS to a final working concentration of 0.1 μg/mL. Stain for 5–20 minutes at room temperature as appropriate, rinse in PBS, mount using an aqueous medium, and examine by fluorescence microscopy. The source notes that suspended cells can be mounted directly in PBS containing DAPI and that similarly treated fixed tissue cells can give comparable staining. The original description states that normal nuclei fluoresce strongly while the cytoplasm does not.
1) Cell scratch assay
Dissociate cells and seed at the source-stated 80% density in 24-well plates, with three replicates per group. After overnight culture, make a horizontal scratch through the center of each well using a presterilized fine bamboo stick or pipette tip, keeping scratch areas comparable. Wash away floating cells with PBS and add fresh medium. Photograph microscopically every 24 hours, recording the initial location and revisiting the same area. Use IPP analysis software to measure scratch areas before and after closure and compare closure rates as an indicator of cell motility.
2) Transwell migration assay (Corning 3422) / invasion assay using Matrigel-coated Transwell inserts (BD 354480)
Before the assay, serum-starve cells for 12–24 hours. Dissociate with trypsin, stop digestion and centrifuge at 1,500 rpm for 3 minutes. Remove the supernatant, wash once with PBS, centrifuge, discard supernatant and resuspend in serum-free medium containing 0.1% BSA. Count cells and adjust to 1 × 10^5 cells/mL in the same medium (required cell density varies by cell type). Add 500 μL complete medium to each well of a 24-well plate (the lower-chamber serum concentration varies by cell type); add 200 μL cell suspension inside each Transwell insert. Carefully transfer inserts with forceps into wells containing complete medium, avoiding bubbles. Incubate the plate at 37°C with 5% CO2 for 24 hours (required time varies by cell type); gently remove medium from the upper chamber and wipe away cells with a PBS-moistened swab. Fix cells by immersing the underside in 10% methanol for 30 seconds, rinse in purified water, stain the underside with crystal violet for 20 minutes and rinse until the background is clear. Count cells that crossed the membrane in five randomly selected microscopic fields. Add 100 μL 33% acetic acid to each insert to elute the stain and measure the eluate OD at 570 nm.
Use the Beyotime β-galactosidase staining kit (C0602). Seed pretreated cells in a 24-well plate at the source-stated 40% density. After 3–4 days, remove supernatant, wash once with PBS and fix with 1 mL staining fixative for 15 minutes at room temperature. Remove fixative and wash three times with PBS or HBSS for 3 minutes each. Remove wash solution and add 1 mL working stain per well. Prepare the working stain in polypropylene, not polystyrene, containers. Incubate overnight at 37°C; the source suggests sealing a six-well plate with parafilm or film to prevent evaporation, although it earlier specifies a 24-well plate; verify the intended format. Examine under a light microscope and count dark-blue cells as senescence-positive.
Tissue obtained fresh at surgery or stored frozen was rapidly warmed at 37°C, washed three times with primary-cell culture medium and trimmed to remove fibrous tissue where possible. Cut the tissue into approximately 1 mm3 pieces and distribute them evenly over the bottom of a 75 cm2 culture flask using a bent pipette. Add a small amount of primary-cell culture medium, carefully invert the flask to help tissue adhere to its bottom, and place it at 37°C in a 5% CO2 incubator for 6–8 hours. Once tissue adheres, return the flask upright, add an appropriate amount of medium and continue culture. Observe growth daily, record when cells fill the flask and collect cultured cells for pathological examination.
① Tumor-cell enrichment by enzymatic dissociation: after 15–20 days of tissue culture, tissue fragments, including nonviable or broken tumor cells and fibroblasts, detach. Remove the flask, discard medium and floating fragments, and wash once with 5 mL PBS. Add 1.5 mL of 0.25% trypsin, rock the flask back and forth four or five times to cover cells, and incubate at 37°C for 2 minutes. Examine under an inverted microscope and stop digestion when the first cells round up and detach; the source identifies these as fibroblasts. Remove these cells and digestion solution. Add 5 mL medium, pipette cells into suspension and collect. Centrifuge and discard supernatant, wash the pellet in PBS, centrifuge again and discard supernatant, then resuspend in medium and seed in plates for continued culture.
② Tumor-cell enrichment by mechanical scraping: prepare a sterile scraper by fitting a rubber tip or a small amount of absorbent cotton to the end of stainless-steel wire and autoclaving it. Mark tumor-cell growth areas on the back of the vessel with an indelible pen. Discard medium, insert the sterile scraper and remove unmarked areas under direct or microscopic observation. Rinse one or two times with PBS to remove scraped cells and continue culture; repeat if fibroblasts remain.
1) Flow cytometric sorting
Under sterile conditions, collect surgically excised lung-cancer tissue, wash twice with PBS and remove necrotic tissue and blood vessels using ophthalmic scissors. Soak the tissue for 10 minutes in five volumes of penicillin (1,000 U/mL) and streptomycin (1,000 U/mL), discard the antibiotics and cut tissue into approximately 1 mm × 1 mm × 1 mm pieces. Add five volumes of digestion solution (0.25% trypsin with 0.02% EDTA) and agitate at 37°C for 30 minutes. Stop digestion with five volumes of complete medium containing 10% fetal bovine serum, invert to release cells, pass through a 200-mesh sieve and centrifuge at 1,500 rpm for 5 minutes. Resuspend the pellet in 100 μL cold PBS, add 4 μL rabbit anti-human CD133-FITC monoclonal antibody and incubate at 4°C in the dark for 30 minutes. Sort CD133-positive and CD133-negative cells by flow cytometry (BD FACSAria), then suspend and culture at 37°C in a 5% CO2 incubator. The source refers to serum-free stem-cell and animal-experiment medium without clearly distinguishing the formulations.
2) Magnetic-bead sorting
Digest tissue to obtain cells as above , wash cells with sorting buffer, centrifuge at 800×g for 3 minutes and discard supernatant. Resuspend the pellet in 300 μL sorting buffer, add 100 μL CD133 immunomagnetic beads and incubate at 4°C in the dark for 30 minutes. Add buffer, wash, centrifuge at 800×g for 10 minutes, discard supernatant and resuspend in buffer. Rinse the column with 500 μL buffer and pass the cell suspension through it. Wash the column three times, remove it from the magnetic field, wash with 1 mL buffer and use pump pressure to collect the eluate as CD133-positive cells.
1) Gene / protein arrays
Six days after GeneA shRNA lentiviral infection, lyse cells in TRIzol and use a small portion for qPCR to assess knockdown efficiency. Freeze the remaining samples at −80°C and send them to the array provider (*** company) for *** array analysis. Sample groups: negative control and experimental group.
Normalize results, identify differential signals and perform GO and pathway analyses to select pathways most altered after GeneA knockdown.
1) Yeast two-hybrid screen for proteins interacting with a target gene (A library screen is illustrated; one-to-one validation is also possible.)
PCR-amplify the GeneA cDNA fragment and clone it into pGBKT7 to construct the pGBKT7-GeneA bait plasmid. Plate AH109 transformed with bait separately on SD/−Trp, SD/−Trp/−His, SD/−Trp/−Ade and SD/−Trp/X-α-Gal media to check bait autoactivation of reporter genes. After confirming no autoactivation, perform yeast two-hybrid screening with the bait-transformed AH109 and a human liver cDNA library pretransformed into Y187. Isolate and sequence cDNA from positive clones and compare sequences with a database to identify candidate GeneA-interacting proteins.
2) Co-IP (One-to-one validation is illustrated; screening can also be combined with mass spectrometry.)
Construct expression vectors for the two molecules to be tested (A and B) and cotransfect 293T cells. Harvest 24–48 hours after transfection, add cell lysis buffer containing protease inhibitors and lyse on ice for 30 minutes. Centrifuge lysate at 4 °C at maximum speed for 30 minutes and collect the supernatant. Reserve a small portion for Western blot analysis; add 1 μg anti-GeneA antibody to the remainder and incubate overnight at 4°C with gentle rocking. Wash 10 μL protein A agarose beads three times in lysis buffer, centrifuging at 3,000 rpm for 3 minutes each time. Add the prepared beads to the antibody-incubated lysate and rock gently at 4°C for 2–4 hours to bind the antibody to the beads. After immunoprecipitation, centrifuge at 3,000 rpm for 3 minutes at 4°C, remove supernatant carefully and wash beads three or four times with 1 mL lysis buffer. Add 15 μL 2× SDS loading buffer, boil for 5 minutes and analyze separately by Western blot using antibodies against A and B.
3) GST pull-down
Approach 1: Given GeneA, identify unknown interacting proteins in A549 cells using GST pull-down combined with mass spectrometry.
Construct a GST-tagged GeneA expression vector and transfect 293T cells. After 48 hours, collect and lyse cells and quantify protein. Pretreat A549 lysate with GST–glutathione agarose beads at 4 °C for 2 hours to remove nonspecific binding proteins; centrifuge at 800×g for 5 minutes at 4°C and collect supernatant. Based on the 293T protein concentration, add excess GST–glutathione agarose beads and the pretreated A549 lysate, then incubate overnight at 4°C. Centrifuge for 5 minutes and wash the pellet with 1× PBS containing 0.5% Triton X-100. Add 2× electrophoresis loading buffer, heat in a boiling water bath for 10 minutes, separate by SDS-PAGE using a 10% resolving gel and stain with Coomassie blue.
Excise protein bands for digestion. Rinse bands twice with double-distilled water and add 100 μL freshly prepared destaining solution (50% acetonitrile and 25 mmol/L NH4HCO3), incubate in a 37 °C water bath for 30 minutes until the bands are clear. Dehydrate with 50% acetonitrile until gel pieces turn white, then reduce and alkylate: add 80 μL 10 mmol/L DTT, incubate at 57°C for 1 hour, remove excess DTT, add 80 μL 6 mmol/L IAA and react in the dark for 45 minutes. Use 25 mmol/L NH4HCO3 to swell the gel, dehydrate with 50% acetonitrile and dry in a vacuum dryer. Digest with trypsin for 16 hours, remove peptide-containing supernatant, add 40 μL extraction solution (100% acetonitrile, 5% TFA) to the gel and perform ultrasonic extraction twice. Combine extracts and supernatant and dry by refrigerated centrifugation. The original text specifies 35 L of sample dissolution solution (0.1% formic acid, 50% acetonitrile); the unit requires verification. Centrifuge at 12,000×g for 12 minutes and analyze the supernatant using a Bruker HCT mass spectrometer.
The example describes local Mascot analysis using the IPI_rat_3.26RAT_v3.26 database from Matrix Science. It gives an allowable peptide relative molecular-mass error of 0.6 without specifying a unit, carbamidomethylation of C as a fixed modification, oxidation of M as a variable modification, and a score threshold above 39.
Approach 2: Given GeneA and GeneB, test whether A and B interact using GST pull-down.
Construct GST-tagged GeneA and His-tagged GeneB expression vectors and cotransfect 293 cells. After 48 hours, collect and lyse cells. Wash 50–70 μL GST beads (immobilized glutathione) once with 800 μL PBS plus 1% Triton-100, mix with 293 cell lysate and incubate for 1 hour at 4°C. Centrifuge to collect beads, wash three times with PBS plus 1% Triton-100 and three times with PBS. Add 40 μL 5× loading buffer to dissolve bead-bound proteins, boil for 3 minutes, centrifuge at high speed and analyze by Western blot with GST and His antibodies. The source specifies lysate and empty GST vector as positive and negative controls, respectively.
Transfect GeneA expression vector into 293T cells in a 10 cm dish. Add 37% formaldehyde to a final concentration of 1%, incubate at 37°C for 10 minutes and add glycine to stop crosslinking. Remove medium, wash twice with PBS, collect cells with a scraper into a 15 mL tube, chill and centrifuge, then remove supernatant. Add SDS lysis buffer according to cell quantity, sonicate and centrifuge. Take 300 μL supernatant (freeze the remainder at −80°C) and divide into three 100 μL portions: add GeneA antibody to the experimental portion; leave one without antibody as a control; to the third add 4 μL 5 M NaCl (final NaCl 0.2 M), reverse crosslinks at 65°C for 3 hours and assess sonication by electrophoresis. Add 900 μL ChIP dilution buffer and 20 μL 50× PIC, then add 60 μL protein A agarose/salmon sperm DNA to each. Rotate for 1 hour at 4°C, let settle for 10 minutes at 4°C, centrifuge at 700 rpm for 1 minute and collect supernatant. Reserve 20 μL from each as input. Add 1 μL antibody to one tube and none to the other; rotate overnight at 4°C. Add 60 μL protein A agarose/salmon sperm DNA, rotate at 4°C for 2 hours, settle for 10 minutes and centrifuge at 700 rpm for 1 minute. Remove supernatant, wash the pellet complex in buffer and elute. Add 20 μL 5 M NaCl per tube, mix and reverse crosslinks overnight at 65°C. Add 1 μL RNase A to each tube and incubate at 37°C for 1 hour; add 10 μL 0.5 M EDTA, 20 μL 1 M Tris-HCl (pH 6.5) and 2 μL 10 mg/mL proteinase K and incubate at 45°C for 2 hours. Recover DNA with the specified gel-recovery kit and dissolve the final sample in 100 μL ddH2O. Design primers and identify products by PCR.
Transfect GeneA expression vector into 293T cells, dissociate, centrifuge and remove supernatant. Lyse with polysome lysis buffer, freeze–thaw and centrifuge, then transfer supernatant to a fresh microcentrifuge tube. Place Sigma beads in a 1.5 mL tube on a magnetic rack, remove supernatant and block for 3 hours. Remove blocking solution, resuspend beads in IP solution, divide equally between two tubes and add cell lysate supernatant. Place on a magnetic rack for several seconds; remove a little supernatant as input mRNA, add TRIzol and leave at room temperature. Rotate the remaining bead-containing IP solution at 4°C for 4 hours, place on the rack, remove supernatant and wash beads four or five times in buffer. Add TRIzol to obtain the output fraction. Extract RNA from input and output with chloroform, reverse-transcribe and design primers for PCR identification.
Cell preparation: expand GeneA-knockdown lentivirus-infected cells to the required quantity. Groups: negative control and experimental group.
Use male six-week-old BALB/c nude mice from Shanghai SLAC Laboratory Animal, 10 per group. After one week of acclimatization, dissociate and centrifuge cells to obtain a single-cell suspension, count and suspend the required amount in PBS, and inoculate subcutaneously in the flank. The source specifies 2 × 10^6 cells in 100 μL per mouse. Measure tumor volume at the injection site every five days. Thirty days after inoculation, the source describes intraperitoneal sodium pentobarbital at 80 mg/kg, photography after anesthesia and cervical dislocation for euthanasia. Remove and weigh tumors and photograph them on a blue background. Divide each tumor: fix one part in 4% paraformaldehyde for later pathology and freeze the other at −80°C.
For a metastasis model, the source also describes opening the abdominal cavity, counting and photographing metastatic nodules, and dividing metastases between 4% paraformaldehyde fixation and −80°C storage.
Embed 4% paraformaldehyde-fixed samples in paraffin and prepare sections for immunohistochemistry. Extract total protein from −80°C frozen samples for Western blot analysis.
Collect clinical pathology samples with complete clinical and follow-up information from 200 patients with *** cancer, and group them by the staging written as “TMN” in the source; verify the intended staging terminology. Prepare paraffin blocks from some tissue for tissue microarrays; extract total RNA from other fresh tissue and reverse-transcribe it to cDNA.
Use immunohistochemistry to assess GeneA in *** cancer tissue microarrays. Based on clinicopathological, treatment and outcome data, analyze correlations between GeneA expression and clinical outcomes (including response rate (RR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS)) and other clinicopathological features. The source specifies SPSS 17.0 for statistical analysis.
Use real-time PCR on reverse-transcribed cDNA to determine tissue mRNA expression of GeneA and other downstream molecules. Combine with clinicopathological information to analyze GeneA in *** tumors of different malignancy grades for differences in expression and examine related pathway molecules.
Lifeint provides EV research and application services from sample preparation, isolation and characterization to omics, functional studies, modification and scale-up. It jointly established a biomedical technology centre with Xiamen University School of Pharmaceutical Sciences, built process-development laboratories to GMP standards, and contributed to an EV expert consensus. Its service records cover more than 300 plant species.
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