For the cellular uptake of exosomes (hTERT MSC Exosomes, ATCC, USA), both ASCs and NSCs were seeded with 500 cells per well on the Cell Culture Slides (Corning®, USA). After 3 days cultured in DMEM containing 10% FBS and 1% AA. Before cellular uptake, the concentration, size distribution, and diameter of exosomes were detected by the Nanosight system (NS300, Malvern, UK). The particles concentration and size distribution were measured by nanoparticle tracking analysis (v3.20 software). All exosomes were labeled with 1 mM Vybrant® CM-Dil solution (Invitrogen, USA). Then, the CM-Dil labeled exosomes were incubated with both ASCs and NSCs in serum-free DMEM at 37 °C for 0, 1, 2, 3, 4 h, respectively. After fixed with 4% paraformaldehyde solution (Santa Cruz, USA) and 5% BSA (Sigma, USA), the cells were stained with F-actin Alexa Fluor™ 488 Phalloidin (Invitrogen, A12379, USA) and Hoechst 33,342 (Thermo, USA) respectively. Finally, the fluorescent images were observed and captured using confocal laser scanning microscopy (LSM 780, Zeiss, Germany). All red area was imported into Image J (v1.48) for quantization.
Paraformaldehyde solution
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Paraformaldehyde solution is a colorless, aqueous solution used as a fixative and preservative in various biological and chemical applications. It is a polymer of formaldehyde that is stable in water. The solution is commonly used in histology, cell biology, and molecular biology techniques to preserve and maintain the structural integrity of cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (4 mentions)
Lsm 780, by Zeiss (3 mentions)
Vybrant cm dil solution, by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Cell culture slides, by Corning (2 mentions)
F actin alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Eclipse ti confocal microscope, by Nikon (2 mentions)
Parkin, by Abcam (2 mentions)
Cox 4, by Santa Cruz Biotechnology (2 mentions)
Rompun, by Bayer (2 mentions)
Tele pack pal 20043020, by Storz (2 mentions)
Red blood cell lysing solution, by BioLegend (2 mentions)
Secondary anti mouse igg antibody, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Fluoview fv10i confocal microscope, by Olympus (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Collagen type 4, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Lonza (1 mentions)
Fluorescent neutravidin beads, by Thermo Fisher Scientific (1 mentions)
29 protocols using paraformaldehyde solution
1
Exosome Uptake in Stem Cells
2
Culturing and Fixation of Cell Lines
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HeLa and SH-EP1 cells were obtained from the American Type Culture Collection, and SH-EP1_α4β2 cells were provided as a gift from Dr. Jie Wu39 (link). The cells were cultured in Dulbecco’s modified eagle medium (Lonza) with 10% fetal bovine serum (Invitrogen) and 1% penicillin and streptomycin in a humidified incubator at 37°C with 5% CO2. The cells were harvested at 75% confluence, transferred to glass or gold-coated glass chips, and cultured overnight before experiments. The glass and gold surfaces were pretreated with 0.3 mg/mL collagen type IV (Sigma-Aldrich) to improve cell attachment to the surface. For experiments using fixed cells, the cells were fixed with 4% paraformaldehyde solution (Santa Cruz Biotechnology) for 20 min, washed with PBS, and immediately placed on an instrument for measurement.
3
Decellularized ECM Modulates Immune Response
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MSCVIVIT+ and MSCVIVIT- (3 x 104/well) were seeded in a 24-well plates and cultured for 7 days in presence of zymosan (1 μg/mL). Media was changed every other day and, in case of MSCVIVIT+, supplemented with doxycycline (0.125 μg/mL). After 7ddays, wells were rinsed with PBS and decellularization was performed using 0.5% Triton + 20 mmol/L NH4OH for 5 min. Matrices were carefully washed with PBS and recellularized with freshly isolated PBMCs (1 x 106/well). Stimulation of PBMCs with zymosan (10 μg/mL) was performed overnight. The supernatants were collected and immediately frozen. IL-8 expression levels in the cell supernatants were assessed using human IL-8/CXCL8 DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA), as per the manufacturer’s instructions.
To verify the integrity of the matrices upon decellularization, some decellularized wells as well as control wells were fixed using 4% paraformaldehyde solution (Santa Cruz Biotechnology), blocked using 2.5% BSA in PBS, and stained with Fibronectin F7387-.2ML (Sigma/Aldrich) and DAPI. Images were then acquired using a fluorescent microscope.
To verify the integrity of the matrices upon decellularization, some decellularized wells as well as control wells were fixed using 4% paraformaldehyde solution (Santa Cruz Biotechnology), blocked using 2.5% BSA in PBS, and stained with Fibronectin F7387-.2ML (Sigma/Aldrich) and DAPI. Images were then acquired using a fluorescent microscope.
4
Immunofluorescence Imaging of Platelet Proteins
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Washed platelets (1 × 106 cells/ml) were allowed to settle on glass‐bottomed dishes for 1 h prior to fixing with 4% paraformaldehyde solution (Santa cruz Biotechnology). The platelets were then washed 2 × 5 min in PBS and permeabilized for 5 min in 0.25% Triton X‐100/PBS. They were blocked for 60 min in 10% bovine serum albumin (BSA)/PBS at 37°C and incubated in 3% BSA/PBS/primary antibody for 2 h at 37°C, or overnight at 4°C, and then washed 6 × 2 min in PBS, followed by an additional incubation for 45 min at 37°C in secondary antibody/3% BSA/PBS. Antibodies for LC3 (Cosmo bio, Japan), Parkin (Abcam, USA), LAMP1 and pp53 (Ser15) (Cell Signal, USA), and CoxIV (Santa Cruz, USA) were used. The stained platelets were observed using a Nikon Eclipse‐Ti confocal microscope with 100× oil immersion lens.
5
THP-1 Phagocytosis Assay of Polysaccharide-Coated Beads
6
Quantifying Cancer Cell Colony Formation
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Cells were harvested and counted by TC20 Automated Cell Counter as described before. A total of 1,000 viable cancer cells in each group were seeded in six-well plate and cultured with 2 ml medium. Three replicates were set for each treatment condition. The culture medium with or without paclitaxel was replaced with fresh medium every 3 days until the formed colonies were clearly visible for naked eyes. Eventually, colonies were washed with PBS, fixed with 4% paraformaldehyde solution (Santa Cruz, sc-281692), and stained by 0.05% crystal violet (Sigma-Aldrich, V5265). The final images were photographed and the ImageJ software was used to analyze the number of colonies in each well.
7
Detailed Endoscopic and Histological Scoring of Murine Colitis
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Prior to endoscopic assessment, the animals were anesthetized intraperitoneally with a mixture of 90–120 mg ketamine (Narketan 10%, Vétoquinol AG, Bern, Switzerland) and 8 mg xylazine (Rompun 2%, Bayer, Zürich, Switzerland) per kg body weight, and examined with the Tele Pack Pal 20043020 (Karl Storz Endoskope, Tuttlingen, Germany). Mice were scored with a MEICS [54 (link)]. For the assessment of the histological scores, 1 cm of the distal third of the colon was removed, fixed in paraformaldehyde solution (4% in PBS, Santa Cruz Biotechnology, Santa Cruz, CA, USA), embedded, stained with hematoxylin and eosin (HE), and scored as described [55 (link),56 (link)].
8
Detailed Endoscopic and Histological Scoring of Murine Colitis
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Prior to endoscopic assessment, the animals were anesthetized intraperitoneally with a mixture of 90–120 mg ketamine (Narketan 10%, Vétoquinol AG, Bern, Switzerland) and 8 mg xylazine (Rompun 2%, Bayer, Zürich, Switzerland) per kg body weight, and examined with the Tele Pack Pal 20043020 (Karl Storz Endoskope, Tuttlingen, Germany). Mice were scored with a MEICS [54 (link)]. For the assessment of the histological scores, 1 cm of the distal third of the colon was removed, fixed in paraformaldehyde solution (4% in PBS, Santa Cruz Biotechnology, Santa Cruz, CA, USA), embedded, stained with hematoxylin and eosin (HE), and scored as described [55 (link),56 (link)].
9
Multicolor Flow Cytometry Staining
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Fluorochrome-conjugated antibodies were added to 100 µl blood at pre-optimized concentrations and incubated for 15 min at room temperature in the dark. Red cells were lysed using red blood cell lysing solution (Biolegend, USA), according to manufacturer's recommendations and cells were washed twice with FACs buffer. Cells were fixed for 15 min in 1% paraformaldehyde solution (Santa Cruz Biotechnology, USA) prior to flow cytometric analysis.
10
Micromass Chondrocyte Differentiation Assay
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In order to induce chondrocyte-like differentiation, micromass cultures were obtained as described by Denker et al., 1999 [3 (link)]. Briefly, cells were trypsinized and 107 cells/ml were resuspended in Ham’s F12 medium with 10% fetal bovine serum. 10 µl drop of cell suspension was placed in the center of a well in a standard 24-well plate (Euroclone® S.p.a). The cells were allowed to adhere for 2–3 h at 37 °C and 5% CO2, and then 500 µl of medium containing 100 ng/ml of BMP2 (Peprotech) was added to each well. Medium was replaced every 3 days.
To check chondrocyte differentiation, cells were either processed for RNA extraction or for Alcian Blue staining to verify glycosaminoglycans deposition. To this aim, micromass cultures were rinsed with Dulbecco’s phosphate-buffered saline (DPBS without Ca and Mg, Euroclone® S.p.a), fixed for 10 min in 4% Paraformaldehyde solution (Santa Cruz Biotechnology, Inc.) and stained overnight with a mixture containing 0.05% w/v Alcian Blue 8GX (Sigma-Aldrich), 0.2 M sodium acetate buffer, pH5.8 and 0.5 M MgCl2.
To check chondrocyte differentiation, cells were either processed for RNA extraction or for Alcian Blue staining to verify glycosaminoglycans deposition. To this aim, micromass cultures were rinsed with Dulbecco’s phosphate-buffered saline (DPBS without Ca and Mg, Euroclone® S.p.a), fixed for 10 min in 4% Paraformaldehyde solution (Santa Cruz Biotechnology, Inc.) and stained overnight with a mixture containing 0.05% w/v Alcian Blue 8GX (Sigma-Aldrich), 0.2 M sodium acetate buffer, pH5.8 and 0.5 M MgCl2.
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