To study the dynamics of immune cells after SCI, spinal cords from sham (laminectomy only) and injured LV-GFP or LV-ChABC treated rats were harvested at day 1, 3, 7, 14 and 28 days after lesion. Animals were deeply anaesthetised with sodium pentobarbital (Euthatal®, 80 mg/kg, administered intraperitoneally) and transcardially perfused with ice-cold 1X phosphate buffered saline (PBS) + 2% EDTA. Immediately after perfusion, 8 mm of the injured spinal cord centred around the lesion epicentre was dissected and placed into ice-cold PBS. Tissue was mechanically dissociated and then passed through a 70 μm cell strainer (BD Falcon, Germany), and centrifuged at 300 × g at 4 °C. The pellet was incubated with Myelin Removal Beads II (Miltenyi Biotec, Germany) and passed through LS columns (Miltenyi Biotech) to elute cells. For primary cell cultures, cells were detached by incubating with Enzyme Free cell dissociation buffer (Milipore). Remaining adherent cells were gently scraped and passed through a 70 μm cell strainer (BD Falcon, Germany). After centrifugation, cells were resuspended in DPBS (Gibco).
Enzyme free cell dissociation buffer
Manufactured by Merck Group
Sourced in United States
Enzyme-free cell dissociation buffer is a laboratory reagent used to gently dissociate adherent cells from cell culture surfaces without the use of enzymes. It is designed to maintain cell viability and minimize disruption to cell surface proteins during the cell passaging process.
Automatically generated - may contain errors
Lab products found in correlation
To pro 3, by Thermo Fisher Scientific (2 mentions)
Polycarbonate membrane, by BD (2 mentions)
Matrigel, by BD (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Myelin removal beads 2, by Miltenyi Biotec (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Vybrant dyecycle violet, by Thermo Fisher Scientific (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Crl 2593, by American Type Culture Collection (1 mentions)
Mc3t3 e1, by American Type Culture Collection (1 mentions)
Dm il led 3, by Leica Microsystems (1 mentions)
Soluble rankl, by Oriental Yeast (1 mentions)
M csf, by R&D Systems (1 mentions)
Osteo assay surface, by Corning (1 mentions)
M csf, by Oriental Yeast (1 mentions)
Lumi4 tb, by PerkinElmer (1 mentions)
Prestoblue, by Thermo Fisher Scientific (1 mentions)
Collagenase type 5, by Merck Group (1 mentions)
11 protocols using enzyme free cell dissociation buffer
1
Immune Cell Dynamics After Spinal Cord Injury
2
Quantifying IgSF11 and VISTA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In total 293 T cells transfected with expression vectors encoding IgSF11 (Flag-tagged at the N-terminus) or VISTA (Flag-tagged at the C-terminus) were washed with PBS and detached using enzyme-free cell dissociation buffer (Millipore). After preparing single-cell suspensions, cells were incubated with the indicated concentration of IgSF11-Fc or control human IgG for 30 min on ice. After washing, the cells were stained with PE-labeled goat anti-human IgG for 15 min on ice. After washing again, cells were stained with TO-PRO-3 (Life Technology, Inc.) to exclude dead cells and analyzed using an LSR (BD Bioscience) analyzer. Surface expression of IgSF11 and VISTA was confirmed using anti-Flag and anti-VISTA antibodies, respectively.
3
Murine Osteoclast Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
dTg-BMMs stimulated with RANKL for the indicated times in the presence of M-CSF were washed with PBS and detached using enzyme-free cell dissociation buffer (Millipore) at 37 °C for 5 min. After generation of single-cell suspensions, cells were counted and suspended at 1 × 106/ml in phenol red-free α-minimal essential medium containing 2% FCS and 2 mm EDTA in polystyrene tubes. Then the cells were stained with 5 μm Vybrant DyeCycle Violet (Life Technologies, Inc.) at 37 °C for 60 min. Finally, the cells were stained with TO-PRO-3 (Life Technologies, Inc.) to exclude dead cells and analyzed using an LSR (BD Biosciences). Excitation laser lines and emission filters were as follows: Vybrant DyeCycle Violet: excitation, 405-nm laser line, and emission, 450/50 BP; mAG: excitation, 488-nm laser line, and emission, 515/20 BP;mKO2: excitation, 532-nm laser line, and emission, 585/42 BP; TO-PRO-3: excitation, 640-nm laser line, and emission, 660/20 BP. Data were analyzed using the FlowJo software (Tree Star).
4
Quantifying Proliferation in BMMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wild Type-BMMs were cultured with M-CSF (60 ng/ml) in the presence or absence of RANKL (150 ng/ml) or cultured with IL-4 (10 ng/ml) plus IL-3 (100 ng/ml) for the indicated amount of time (in hours). BrdU (10 μm ) were added to the culture for the last 6 h. After culture, cells were washed with PBS and detached using enzyme-free cell dissociation buffer (Millipore) at 37 °C for 5 min. After generation of single-cell suspensions, cells were counted and suspended at 1 × 106/ml in PBS in polystyrene tubes. The cells were stained with LIVE/DEAD Aqua (Life Technologies, Inc.) for 30 min on ice to exclude dead cells. Cells were washed with PBS with 2% FCS and fixed with BrdU fixation buffer (eBioscience) for 1 h on ice followed by DNase treatment. Cells were stained with FITC-labeled anti-BrdU antibody (clone Bu20a, eBioscience) for 20 min, and after washing, cells were further stained with 7-AAD. Finally, the cells were analyzed using a FACSCanto II (BD Biosciences). Numbers indicate the percentages of S phase cells. Results are representative of three independent experiments.
5
Boyden Chamber Cell Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell migration was assessed as previously described [60 (link)]. Briefly, MCF7 cells were serum-starved overnight. Next day cells were detached with enzyme-free cell dissociation buffer (Millipore) and 1.5 × 105 cells resuspended in serum-free DMEM. The polycarbonate membranes (8 μm pores, BD Bioscience) of inserts were coated with high concentration Matrigel (BD Bioscience) diluted in serum-free DMEM (1:1000). Cells were placed in the inner compartment of Boyden chamber inserts and allowed to migrate for 24 h toward DMEM (10% FBS) ± 50 ng/ml FGF7 and/or 100 nM Pg. Non-migrated cells were removed by cotton swab. Membranes were mounted onto glass slides, cells stained with DAPI and counted in 20 random fields (100x) under AxioVert 200 fluorescent microscope.
6
Transwell Cell Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell migration was assessed as previously described (30 (link)). Briefly, indicated cell lines were seeded onto 6-cm plates. Next day, cells were either pre-treated with CPL304110 (1 μM) or PF431396 (100 nM) for 2 h. Subsequently, cells were detached with enzyme-free cell dissociation buffer (Millipore) and 2.5 × 104 cells were resuspended in serum-free medium ± indicated inhibitor. The polycarbonate membranes (8 μm pores, BD Bioscience) of inserts were coated with high concentration Matrigel® (BD Bioscience) diluted in serum-free medium (1:1). Cells were placed in the inner compartment of the Boyden chamber inserts and allowed to migrate towards complete medium (10% FBS) ± indicated inhibitor. After 24 h of incubation, the non-migratory cells were removed using cotton swab. Membranes were mounted on to glass coverslips, migratory cells were stained with DAPI and counted from 20 random fields under AxioVert 200 fluorescent microscope.
7
In vitro Osteoblast Differentiation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro osteoblast differentiation has been described previously19 (link),46 (link). Briefly, bone marrow cells (5.3 × 105 per cm2) were collected by flushing long bones of mice and maintained for 3 days in α-minimum essential medium (α-MEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C in a humidified incubator containing 5% CO2. The cells were maintained in osteogenic medium (α-MEM, 10% FBS, 1% penicillin–streptomycin, 50 mg/mL ascorbic acid, and 10 mM beta-glycerophosphate) for 14 days to promote osteoblastic differentiation and mineralization. The osteogenic medium was refreshed every 3 days.
MC3T3-E1, a clonal osteoblastic cell line derived from newborn mouse calvaria, was purchased from the American Type Culture Collection (CRL-2593™: ATCC, Manassas, VA, USA). Cells (1.05 × 104 per cm2) were maintained for 2 days in α-MEM containing 10% FBS and 1% penicillin–streptomycin. For osteoblastic differentiation and mineralization, cells were maintained in osteogenic medium, which was refreshed every 3 days.
To generate BMMs, non-adherent bone marrow cells from C57BL/6J mice were seeded in a 10 cm Petri dish and cultured in α-MEM containing 10% FBS, 1% penicillin–streptomycin, and 10 ng/mL M-CSF. After 3 days, adherent cells were collected in enzyme-free cell dissociation buffer (Millipore, Burlington, MA, USA) at 37 °C and used as BMMs.
MC3T3-E1, a clonal osteoblastic cell line derived from newborn mouse calvaria, was purchased from the American Type Culture Collection (CRL-2593™: ATCC, Manassas, VA, USA). Cells (1.05 × 104 per cm2) were maintained for 2 days in α-MEM containing 10% FBS and 1% penicillin–streptomycin. For osteoblastic differentiation and mineralization, cells were maintained in osteogenic medium, which was refreshed every 3 days.
To generate BMMs, non-adherent bone marrow cells from C57BL/6J mice were seeded in a 10 cm Petri dish and cultured in α-MEM containing 10% FBS, 1% penicillin–streptomycin, and 10 ng/mL M-CSF. After 3 days, adherent cells were collected in enzyme-free cell dissociation buffer (Millipore, Burlington, MA, USA) at 37 °C and used as BMMs.
8
Osteoclast Differentiation and Resorption Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nonadherent bone marrow cells derived from mice were seeded (1.5–2.0× in a 10 cm Petri dish) and cultured in the presence of 30 ng/mL M-CSF (R&D Systems, Minneapolis, MN, USA). After 3 days, adherent cells were collected in enzyme-free cell dissociation buffer (Millipore, Burlington, MA. USA) at 37 °C and used as bone marrow macrophages (BMMs). BMMs were further cultured in the presence of 50 ng/mL soluble RANKL (Oriental Yeast Co., Ld., Tokyo, Japan) and 10 ng/mL M-CSF to generate osteoclasts70 (link). Three days later, TRAP staining was conducted using an Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma-Aldrich). TRAP+ multinucleated (>3 nuclei) cells were regarded as mature osteoclasts.
To evaluate bone resorptive activity, osteoclasts were cultured on RepCell dishes for 5 days. Next, the cells were transferred to an Osteo Assay Surface (Corning, Corning, NY, USA). After 48 h, resorption pits were observed under an inverted light microscope (DM-IL-LED-3; Leica Microsystems), and the binarized areas of resorption pits were measured using NIS Elements integrated software (Nikon, Tokyo, Japan).
To evaluate bone resorptive activity, osteoclasts were cultured on RepCell dishes for 5 days. Next, the cells were transferred to an Osteo Assay Surface (Corning, Corning, NY, USA). After 48 h, resorption pits were observed under an inverted light microscope (DM-IL-LED-3; Leica Microsystems), and the binarized areas of resorption pits were measured using NIS Elements integrated software (Nikon, Tokyo, Japan).
9
SNAP-tagged Protein Fluorescence Labeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SNAP-tagged proteins (ACE2, LepR, VEGFR2) were fluorescently labeled by incubating cells transfected with the corresponding expression vectors with an SNAP suicide substrate conjugated to the long-lived fluorophore Terbium cryptate (Tb; Lumi4-Tb, 100 nM; Cisbio Bioassays) in Tag-lite labeling medium (1 h, on ice) (Keppler et al., 2003 (link)). After several washes, cells were collected using enzyme-free cell dissociation buffer (Sigma-Aldrich), resuspended in Tag-lite buffer and distributed into a 384-well plate. Efficient fluorescent labeling of SNAP was verified by reading fluorescence signal at 620 nm. Experiments were then conducted according to the different modes described below. All reagents were diluted in Tag-lite buffer (final reaction volume of 14 μL) and incubations were performed at room temperature; except in the case of compounds Fenbendazole and Elaidic Acid, where incubation was performed at 37°C to avoid precipitation of compounds. TR-FRET signals were detected using a plate reader (Tecan F500; Tecan, Männedorf, Switzerland) with the following settings: excitation at 340 nm (Tb, energy donor), emission at 665 nm (d2, acceptor) and 620 nm (donor); delay of 150 μs; and integration time of 500 μs. Data is expressed as TR-FRET ratio (acceptor/donor). When indicated, TR-FRET ratio was normalized to % of basal or % of maximal binding (Bmax).
10
Serotonin-Induced Cell Adhesion Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hu2AAB1 cells and HEK293 cells were harvested using an enzyme free cell dissociation buffer (Sigma, USA), re-suspended in DMEM containing dialyzed serum (which is serotonin-free) supplemented with 10 μM serotonin, and the cell suspension was diluted to 1.5 × 105 cells/ml. Then 20 μM RGD peptide was added to the suspension and incubated at 37 °C and 5% CO2 for 60 min. Following the incubation, 6 × 104 cells were plated onto fibronectin pre-coated (0.1 μg/ml, 60 min at 37 °C) wells of a 24-well dish and allowed to attach for 60 min. Finally, the media along with the unattached cells were aspirated and the attached cells were counted using PrestoBlue (Invitrogen, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!