The air–liquid interface (ALI) culture of stem cell clones was performed as described [12 (link)]. The colonies were digested in a 0.25% trypsin–EDTA solution at 37 °C for 8 min and passed through 30 µm filters (Miltenyi Biotec) to obtain single cells. The pellets were resuspended in 80 μL F12 and 20 μL mouse feeder removing beads (Miltenyi Biotec) and incubated at 4 °C for 15 min to avoid light. Each Transwell insert (Corning) was coated with 20% Matrigel (growth factor reduced, BD Biosciences) and incubated at 37 °C for 30 min to polymerize. 200,000 feeder cells were seeded into each Transwell insert and incubated overnight at 37 °C in 7.5% CO2. Placed columns in the magnetic MACS Separator and rinsed with 3 mL F12 buffer. Then 400 μL F12 to the cells were added before passing through the columns. Finally, colonies were eluted with another 2 mL F12. 200,000—300,000 cells were seeded into each Transwell insert and cultured with SCM medium. After reaching confluency (3—7 days), the top medium was removed by carefully pipetting, and the cells were further incubated for 6—12 days in SCM minus nicotinamide medium (SCM-6) before analysis. The SCM-6 medium was changed every day.
30 m filter
Manufactured by Miltenyi Biotec
Sourced in Germany
The 30 µm filter is a lab equipment designed to separate particles or cells based on size. It is a membrane filter with a pore size of 30 micrometers, allowing the passage of smaller components while retaining larger ones.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (1 mentions)
Transwell insert, by Corning (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Aria sorp cell sorter, by BD (1 mentions)
Anti biotin microbead, by Miltenyi Biotec (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Guava easycyte benchtop flow cytometer, by Merck Group (1 mentions)
Cyto fast fix perm buffer, by BioLegend (1 mentions)
C tube, by Miltenyi Biotec (1 mentions)
Gentlemacs, by Miltenyi Biotec (1 mentions)
Mouse neutrophil isolation kit, by Miltenyi Biotec (1 mentions)
70 μm filter, by Miltenyi Biotec (1 mentions)
Murine m csf, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Papain, by Worthington (1 mentions)
Facscalibur system, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
9 protocols using 30 m filter
1
Air-Liquid Interface Stem Cell Cloning
2
Cell Isolation and Sorting Protocol
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When the colonies were generated after processing tissue, cells were digested in a 0.25% trypsin‐EDTA solution (Gibco) for 5 to 8 min at 37 °C and cell suspensions were passed through 30 µm filters (Miltenyi Biotec). 106 cells were blocked with 0.1% FBS at 4 °C for 30 min, then incubated with CD326 Monoclonal Antibody (Thermo Fisher Scientific, 53‐9326‐42, 1:50) at 4 °C for 30 min. Samples were collected and sorted with an Aria SORP Cell Sorter (BD). Single cells were seeded into 96‐well plates coated with feeder cells.
3
Isolation of Satellite Cells from Injured Mouse Muscles
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TA muscles were isolated from C57BL/6J mice at time zero (uninjured controls), and 3 and 10 d after freeze injury. The muscles were dissected and finely chopped with scissors and incubated with 0.2% Collagenase in DMEM for 30 min at 37°C while gently shaking (250 rpm). This step of the procedure was performed twice. The cells were then filtered with 30-µm filters (Miltenyi Biotec). 1 ml of serum was added and the suspension was centrifuged for 20 min at 1,200 rpm at 4°C. The supernatant was decanted and the staining was performed with the SM/C-2.6 antibody (Fukada et al., 2004 (link); 1:200 for 40 min on ice) and with anti-Biotin MicroBeads (Miltenyi Biotec) as secondary antibody according to manufacturer’s protocol. After magnetic sorting the cells were centrifuged 20 min at 2,500 rpm and the pellet was snap frozen on dry ice.
4
Profiling Lung Organoid Cell Types
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Lung organoids were dissociated into single cells via trypLE digestion and strained with a 30 µm filter (Miltenyi Biotec, Germany). Approximately 2.5E5 cells for each sample were fixed and permeabilized at room temperature in Cyto-Fast Fix Perm buffer (BioLegend, USA) for 20 min. The samples were subsequently washed with Cyto-Fast Perm Wash solution (BioLegend) and incubated with lung epithelial cell-type markers for 30 min. Following primary antibody incubation, the samples were washed and incubated with propidium iodide (Invitrogen) and Alexa Flour 488 secondary antibodies (Invitrogen) for 30 min. Samples were resuspended in FACS buffer (PBS, 5% FBS, 2 mM sodium azide). Flow cytometry was performed using Guava easyCyte Benchtop Flow Cytometer (Millipore) and data was analyzed using InCyte (version 3.3) and FlowJo X v10 software.
5
Mouse Spleen Isolation and Characterization
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Mice were sacrificed by cervical dislocation. Spleen was removed and a single-cell suspension was obtained using gentleMACS, Miltenyi (program: m-spleen-01) with C-tubes (Miltenyi) in PBS. Next, single-cell solution was filtered using 100 µM cell strainers (Thermo Fischer). GEYS solution was used for erythrocyte lysis for 2 min at 4°C. After erythrocyte lysis cells were suspended in supplemented RPMI 1640 and filtered through a 30 µM filter (Miltenyi). Cell number was determined using a Neubauer chamber. All steps were performed at 4°C. All centrifugation steps were performed at 4°C, 300 g, 7 min.
6
Bone Marrow Cell Isolation
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Mice were sacrificed by cervical dislocation. One tibia and femur were excised per mouse. Femur and tibia were flushed with supplemented RPMI 1640 and erythrocytes were lysed using GEYS solution (2 min at 4°C). After erythrocyte lysis, cells were suspended in supplemented RPMI 1640 medium and filtered through a 30 µM filter (Miltenyi). Cell numbers were determined using a Neubauer chamber. All steps were performed at 4°C. All centrifugation steps were performed at 4°C, 300 g, 7 min.
7
Isolation and Culture of Mouse Bone Marrow Cells
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To isolate whole bone marrow cells, long bones of 8–10-week-old C57BL/6J mice were triturated with mortar and pestle in PBS supplemented with 0.5% bovine serum albumin and 2 mM EDTA. Cells were filtered through a 70-μm filter (Miltenyi Biotech), washed, and centrifuged (10 min; 300 g; 4 °C). After erythrocyte lysis in hypotonic NH4Cl − buffer, cells were filtered through a 30-µm filter (Miltenyi Biotech). For neutrophil isolation the mouse Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec) was used according to the manufacturer’s instructions. Bone marrow derived non-neutrophils that were retained in the separator column after neutrophil isolation were plated in non-tissue culture treated dishes and treated with 20 ng/µl murine M-CSF (315-02, Peprotech) for 7 days to generate macrophages56 (link).
8
Single Cell Isolation for Transplantation
9
Quantifying GFP Cells in Retinal Organoids
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Quantification of GFP positive cells in retinal organoids was performed in 3 biological replicates, meaning that each sample of 10 organoids was obtained from 3 different protocols of differentiation. Each sample was dissociated using two units of pre-activated papain at 28.7 u/mg (Worthington) in Ringer solution for 25 min at 37 °C. Once a homogeneous cell suspension was obtained by repeated pipetting, papain was deactivated with Proneural medium (Table 1 ). Cells were filtered with 30 µm filter (Miltenyi, Bergisch Gladbach, Germany) and fixed for 10 min at 4 °C with 4% paraformaldehyde. Cells were washed in PBS. Samples were analysed by flow cytometry and at least 10,000 events were analysed in each experiment using FACSCalibur system (BD Biosciences, Allschwil, Switzerland). The number of positive cells within the gated population was analysed using CellQuest™ Pro (BD Biosciences) software. Non-infected organoids serve as controls.
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