After transfection, A549, PC-9 (2 × 102/well) and Calu-1 cells (7 × 102/well) were cultured in 6-well plates for 14 days. Paraformaldehyde fix solution (4% PFS) and 0.1% crystal violet (Beyotime Biotechnology) were added into 6-well plates. Visible cell colonies were then counted.
Paraformaldehyde fix solution
Manufactured by Beyotime
Sourced in China
Paraformaldehyde Fix Solution is a laboratory reagent used for the fixation and preservation of biological samples. It is a 4% solution of paraformaldehyde in phosphate-buffered saline (PBS). This solution is commonly used in various biological and biomedical applications, including histology, immunohistochemistry, and cell biology, to maintain the structural integrity and morphology of cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet staining solution, by Beyotime (7 mentions)
Crystal violet, by Beyotime (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Immunostaining permeabilization buffer with triton x 100, by Beyotime (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Lsm 710, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Antifade mounting medium with dapi, by Beyotime (2 mentions)
Dapi staining solution, by Beyotime (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 cck 8, by Beyotime (2 mentions)
Dxp athena flow cytometer, by Cytek (1 mentions)
Accutase solution, by STEMCELL (1 mentions)
Transwell, by Beyotime (1 mentions)
Matrigel, by BD (1 mentions)
Transwell, by BD (1 mentions)
Transwell, by Corning (1 mentions)
Dab substrate kit, by Solarbio (1 mentions)
Ht10132 1l, by Merck Group (1 mentions)
41 protocols using paraformaldehyde fix solution
1
Cell Colony Formation Assay
2
Macrophage Staining and Flow Cytometry Analysis
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The macrophages were washed with DPBS and then incubated with Accutase solution (STEMCELL Technologies) for 15 min at 37 °C. Following incubation, macrophages were detached by robust pipetting, and then the cells were collected and washed with PBS/BSA (PBS + 2% BSA). For surface staining, the cells were directly incubated with antibodies against surface markers. For intracellular staining, the cells were fixed with 4% paraformaldehyde fix solution (Beyotime Biotechnology) and permeabilized with immunostaining permeabilization buffer with Triton X-100 (Beyotime Biotechnology). The cells were then incubated with antibodies against intracellular proteins. At least 5000 cells per sample were analyzed. Samples were acquired with a DxP Athena flow cytometer (Cytek Biosciences, Fremont, CA, USA) and analyzed using FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA). Background signals were controlled by applying a fluorescence minus one (FMO) control.
3
Evaluating Cell Migration and Invasion
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To measure cell migration, scratch wounds were generated on the surface of 24-well plates by straightly moving a 100 μL of pipette across the center of every well. The remaining 5 × 104 cells (H1299, A549, or H460) were cultured in serum-free medium, and wounds were photographed after wound healing for 0 and 24 h. Wound area was measured on ImageJ v1.52 software (National Institute of Health, Bethesda, MD, USA), and the percentage of wound closure was calculated.
Transwell (Corning, Corning, NY, USA) was utilized to measure cell invasion. First of all, Transwell supports were coated with Matrigel (BD Biosciences); then, 5 × 104 H1299, A549, or H460 cells were collected in serum-free medium and seeded in the upper chamber, and the lower chamber was filled with medium containing 10% fetal bovine serum. Transwell invasion assay was incubated at 37°C for 48 h, and invaded cells on the lower surface were fixed with 4% Paraformaldehyde Fix Solution (Beyotime) and stained with Crystal Violet Staining Solution (Beyotime) for 20 min. The stained cells were observed under a microscope (100×; Olympus, Tokyo, Japan).
Transwell (Corning, Corning, NY, USA) was utilized to measure cell invasion. First of all, Transwell supports were coated with Matrigel (BD Biosciences); then, 5 × 104 H1299, A549, or H460 cells were collected in serum-free medium and seeded in the upper chamber, and the lower chamber was filled with medium containing 10% fetal bovine serum. Transwell invasion assay was incubated at 37°C for 48 h, and invaded cells on the lower surface were fixed with 4% Paraformaldehyde Fix Solution (Beyotime) and stained with Crystal Violet Staining Solution (Beyotime) for 20 min. The stained cells were observed under a microscope (100×; Olympus, Tokyo, Japan).
4
Colony Formation Assay
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Tumor cells were seeded into 6-well plates for 24 h, with 800–1000 cells/well, and treated with compound 25 at 0, 1, 2.5, or 5 μM for 1–2 weeks. When visible clones appeared on the plates, the colonies were fixed with 4% paraformaldehyde fix solution (Beyotime, Beijing, China) and stained with 0.2% crystal violet (Beyotime, Beijing, China). After the stains were washed and dried, images were photographed, and then processed using Photoshop. Colonies were quantified using ImageJ V1.8.0.
5
Histological Analysis of Mouse Testes and Epididymis
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Cauda epididymis and testes from adult mice were fixed in Bouin's solution (Sigma, HT10132‐1L) at 4°C for 24 h, then embedded in paraffin, and cross‐sectioned. Sections mounted on glass slides were dewaxed and stained with haematoxylin and eosin (HE) for cauda epididymis, or with Periodic acid Schiff (PAS) for testes using PAS staining kit (KeyGEN BioTECH, KGA222). For in situ cell death analysis, testes from adult mice were fixed in 4% Paraformaldehyde Fix Solution (Beyotime, P0099), and TUNEL staining was performed using an In Situ Cell Death Detection Kit, POD (Roche, 11684817910) and DAB Substrate kit (Solarbio, DA1010).
6
Tissue Fixation and Histological Analysis
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After tumour dissection, the tissues were fixed with 4% Paraformaldehyde Fix Solution (Beyotime) at 4°C for 6 h. Haematoxylin–eosin (HE) staining, immunohistochemistry (IHC) and IF analyses of tissues were conducted by Servicebio.
7
Clonogenic Assay for X-ray Irradiated Cells
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A certain number of NPC cells were cultured in six-well plates. After cell adherence, transfection or EXO co-culture was performed. The cells were irradiated with X-rays (4Gy or 8Gy) and dosimetry confirmed the central dose rate of 100 cGy/min (X-RAD 225 high-energy biological X-ray irradiator, USA), then further incubated at 37 °C with 5% CO2 for 2-3 weeks. After cell colonies (50 cells) were formed in the six-well plate, the cells were washed twice with phosphate buffered saline (PBS) (Solarbio, P1020), fixed with 4% Paraformaldehyde Fix Solution (Beyotime, P0099) for 10 min, cleaned 2 times with PBS, stained with 0.1% crystal violet (Beyotime, C0121) at 37 °C for 10 min, followed by PBS cleaning twice, and photographed after drying.
8
Cell Migration and Invasion Assays
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Cell migration was assessed by wound healing assay. Cells were grown in 24-well plates. Till they reached 80% confluence, a scratch was made with a 200-μl sterile pipette tip. The wounded closures were imaged at 0 and 24 h after the scratches were made using an inverted microscope. Invasion assay was performed with 24-well BioCoat Matrigel Invasion Chambers (BD) according to the manufacturer’s instructions. The cells that invaded through the Matrigel were first fixed with 4% Paraformaldehyde Fix Solution (Beyotime) for 20 min and then stained with crystal violet (Beyotime) for 15 min at 37 °C. After washing with PBS, 5 randomly selected fields were imaged (×400) and counted.
9
Fluorescence Imaging of Transfected Cells
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Three days after transfection, the cells were washed with 1X Phosphate Buffered Saline with Tween® 20 (PBST, 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, 0.05% (v/v) Tween-20), three times, and fixed in 4 % Paraformaldehyde Fix Solution (Beyotime, Shanghai, China, P0099-100 mL) at 25 °C for 10–15 min. The cells were washed three times with 300 μL 1X PBST. Then, 100 μL of 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA, D1306) working solution were added, and the samples were incubated for 10 min at 25 °C in the dark. The cells were washed again with PBST, three times every 5 min. Finally, the cells were imaged using fluorescence microscope (Olympus, Tokyo, Japan) at different magnifications. The used excitation wavelengths for DAPI, DsRed, and EGFP were 405 nm, 488 nm, and 559 nm, respectively. The cells were also imaged using the confocal microscope (Olympus, FV1000, Tokyo, Japan).
10
Visualizing Nanoparticle Cellular Uptake
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The cellular uptake of the nanoparticles was visualized by Perl’s Prussian blue staining. The cells were cultured in 24-well plates and transfected as indicated. After incubation for 12 h, the cells were fixed with 4% Paraformaldehyde Fix Solution (P0099, Beyotime) for 15 min. The cells were washed with PBS and stained with 5% potassium ferrocyanide in 10% hydrochloric acid for 30 min at room temperature. The working solution was made by mixing equal volumes of 10% potassium ferrocyanide and 20% hydrochloric acid solution just before use. The cells were observed under a microscope (Olympus CKX41, Tokyo, Japan).
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