For flow cytometry of tissue culture cells, cells were isolated from mouse spleens and mechanically dissociated with a 40 µm cell strainer (Greiner Bio-One). Red blood cells were lysed with RBC lysing buffer (Sigma). Cells were labeled with aqua live/dead fixable viability dye in PBS (1:500), followed by surface antibodies (1:100) in staining buffer (1% BSA, 1% rat serum in PBS). Antibodies used for surface staining: PE/Dazzle anti-CD4 (RM4-5, Biolegend), APC/CY7 anti-CD8 (YTS156.7.7, Biolegend), PE/CY7 anti-CD44 (IM7, Biolegend), anti-CD69 (H1.2F3, BD Bioscience). For intracellular staining, cells were stained with aqua live/dead dye, followed by surface staining, fixation with perm/fixation solution (Thermofisher), and stained with intracellular antibodies against FITC anti-IFNγ (XMG1.2, Invitrogen) and AF647 anti-tumor necrosis factor α (TNFα) (MP6-XT22, Biolegend) in 1 x perm/wash buffer. Cells were washed and analyzed on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software.
40 m cell strainer
Manufactured by Greiner
Sourced in Austria
The 40 µm cell strainer is a laboratory equipment designed to filter and separate cells and other biological materials based on their size. It features a mesh with a nominal pore size of 40 micrometers, allowing the passage of smaller particles while retaining larger ones.
Automatically generated - may contain errors
Lab products found in correlation
Rbc lysing buffer, by Merck Group (3 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
100 m cell strainer, by Greiner (2 mentions)
Foxp3 fix perm kit, by Thermo Fisher Scientific (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Pe cy7 anti cd44 im7, by BioLegend (1 mentions)
Mp6 xt22, by BioLegend (1 mentions)
Anti cd69 h1.2f3, by BD (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Facsaria 1, by BD (1 mentions)
Anti cd45 pe, by BD (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Anti cd31 fitc, by BD (1 mentions)
Recombinant murine gm csf, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
12 well suspension culture plates, by Greiner (1 mentions)
Neurocult basal medium, by STEMCELL (1 mentions)
Accutase, by STEMCELL (1 mentions)
Liver dissociation kit, by Miltenyi Biotec (1 mentions)
13 protocols using 40 m cell strainer
1
Multiparametric Flow Cytometry of Immune Cells
2
Sorting and Isolation of Endothelial Cells and Pericytes
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Freshly isolated SVF cells were resuspended in EGM-2 medium, filtered through a 40 µm cell strainer (Greiner, Austria), and cell yield was determined. The 5 × 106 cells were incubated with 25 µL of primary antibodies anti-CD31-FITC (BD Biosciences, Austria), anti-CD146-PerCP (R&D, Germany) and anti-CD45-PE (BD), for 30 min at 4 °C in 250 µL of FACS Buffer (PBS, 0.1% BSA, 0.2 mM Glutamine) or remained unstained to serve as the control. Afterwards, the cells were washed with FACS Buffer, centrifuged at 300× g for 5 min, resuspended in 2.5 mL of FACS Buffer and filtered through a 40 µm cell strainer. Cell sorting was performed on a FACS Aria I (Becton Dickinson, Heidelberg, Germany) using a 100 µm nozzle to yield two distinct cell populations, which were as follows: endothelial cells sorted as CD31+, further referred to as ‘EC’ and pericytes sorted as CD45−/CD31−/CD146+, further referred to as ‘PC’. Unstained cells were used to determine the background autofluorescence. Cell aggregates were excluded in an FSC-W versus FSC-A gate. Sorting was performed in ‘Purity’ mode and approximately 3 × 105 EC and PC were collected in 1 mL of EGM-2. After centrifugation at 300× g for 5 min, the cell pellets were resuspended in 800 µL of TRI Reagent® (Sigma-Aldrich). Until RNA isolation, the samples were stored at −80 °C.
3
Isolation and Culture of Murine Spleen and Bone Marrow Cells
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Spleens were mechanically disrupted using a pestle and a 40 µM cell strainer (Greiner Bio-One, Frickenhausen, Germany) to obtain a single-cell suspension. Spleen cells (2 × 106/500 µL) were cultured in FACS tubes overnight in medium (IMDM, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (all from Sigma-Aldrich, Deisenhofen, Germany) and 50 µM ß-mercaptoethanol (Roth) containing 5% FBS (PAN-Biotech, Aidenbach, Germany)). Bone marrow cells (2 × 105/mL) were seeded in 12-well suspension culture plates (Greiner Bio-One) in culture medium supplemented with recombinant murine GM-CSF (10 ng/mL; Miltenyi, Bergisch Gladbach, Germany). Culture media were replenished on days 3 and 6 of culture.
4
Generating Neurospheres from Mouse Hypothalami
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For each litter, we collected six hypothalami to prepare neurospheres. After one wash in 2 mL of sterile PBS containing 2% glucose, hypothalami were mechanically triturated in 1 mL of NeuroCult Basal Medium (STEMCELL Technologies Inc., Vancouver, BC, Canada) using a 1 mL micropipette until a single-cell suspension was obtained. The cell suspension was then filtered on a 40 µm cell strainer (Greiner Bio-one International GmBH, Kremsmünster, Austria) and centrifuged at 500× g for 5 min. Cell pellets were resuspended in NeuroCult Basal Medium and viable cells were counted on a hemocytometer after eosin staining. All cells from one hypothalamus were seeded in a T-12.5 cm2 tissue culture flask containing 5 mL of Complete NeuroCult TM Proliferation Medium and incubated at 37 °C and 5% CO2. The obtained neurospheres were passed after 3 days in vitro. Briefly, cell passages were done by centrifuging neurospheres at 90× g for 5 min and incubating pellets with 200 µL of Accutase (STEMCELL Technologies Inc.) followed by gentle trituration to obtain single-cell suspensions. Cells were washed in NeuroCult Basal Medium, centrifuged at 500× g for 5 min and resuspended in NeuroCult Basal Medium, Complete NeuroCult TM Proliferation Medium or Complete NeuroCultTM Differentiation Medium, depending on the following experiment.
5
Isolation of Liver and Immune Cells
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Liver nonparenchymal cells (NPC) were isolated using a liver dissociation kit and the gentle MACS dissociator (both Miltenyi Biotec) as recommended by the manufacturer with some modifications. In brief, after liver dissociation, cells were resuspended in 1 mL cold HBSS buffer (w/o Ca2+ and Mg2+). The cell suspension was mixed with a double volume of freshly prepared 30% HistoDenZ (Sigma-Aldrich) and overlaid with 1 mL of cold HBSS buffer. After centrifugation (1500× g, 4 °C, w/o break), the cell containing interphase was retrieved and washed. Erythrocytes were lysed using a hypotonic buffer.
Spleen cells were isolated using a 40 µM cell strainer (Greiner Bio-One) to obtain a single-cell suspension. In parallel settings, spleen cells were resuspended in medium (IMDM, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (all from Sigma-Aldrich, Deisenhofen, Germany), and 50 µM ß-mercaptoethanol (Roth, Karlsruhe, Germany)) containing 5% FCS (PAN).
Bone marrow cells (2 × 105/mL) were seeded in 12-well cell cluster plates (Greiner Bio-One) in an IMDM-based culture medium (see above) supplemented with 10 ng/mL recombinant murine GM-CSF (R&D Systems, Wiesbaden, Germany). Culture media was replenished on days 3 and 6 of culture.
Spleen cells were isolated using a 40 µM cell strainer (Greiner Bio-One) to obtain a single-cell suspension. In parallel settings, spleen cells were resuspended in medium (IMDM, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (all from Sigma-Aldrich, Deisenhofen, Germany), and 50 µM ß-mercaptoethanol (Roth, Karlsruhe, Germany)) containing 5% FCS (PAN).
Bone marrow cells (2 × 105/mL) were seeded in 12-well cell cluster plates (Greiner Bio-One) in an IMDM-based culture medium (see above) supplemented with 10 ng/mL recombinant murine GM-CSF (R&D Systems, Wiesbaden, Germany). Culture media was replenished on days 3 and 6 of culture.
6
Rapid Lung Cell Isolation in Mice
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BALB/c mice were pre-heated at 37 °C in a heat box for 10 min. 2.5 μg of FITC-conjugated anti-CD45.2 (eBiosciences) was injected intravenously into the tail of the mice in a volume of 100 μl. One minute after the injection, mice were sacrificed, and the lungs were collected. Lungs were placed in a C-tube (Miltenyi Biotec) containing the digestion media (RPM1 with collagenase and DNase). A gentleMACS dissociator was used to homogenise the lungs according to the manufacturer’s protocol. The homogenised lungs were incubated in a shaking incubator at 37 °C for 1 h. The content was poured through a 100 µm cell strainer (Greiner Bio-One) into a 50 ml tube. The samples were spun for 8 min, 300 × g at 4 °C. After the spinning, the cells were treated with ACK buffer for 5 min and filtered using a 40 µm cell strainer (Greiner Bio-One) before spinning as above. The cell pellet was resuspended in complete media. To determine total cell numbers, the cells were counted using CASY counter.
7
Generation of Murine Bone Marrow-Derived Dendritic Cells
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Bone Marrow (BM) of C57BL/6J mice was isolated by cutting the ends of femur and tibia and flushing the bone cavities with Phosphate Buffer Saline (PBS, pH 7.4), supplemented with 1% FCS using a 23G cannula. The BM was collected in a 50 mL tube and centrifuged at 1300 rpm for 10 min, followed by lysis of erythrocytes by a 2 min incubation in Greys lysis buffer. The reaction was stopped by adding PBS with 1% FCS. The cell suspension was filtered through a 40 µm cell strainer (Greiner Bio-one) and the obtained cells were resuspended in IMDM culture medium (Iscove’s Modified Dulbecco's Medium, 5% FCS, 2 mM l -glutamine, 1% sodium pyruvate). Cells were seeded in six-well nonadherent plates (3–6 × 106/well) and culture medium was supplemented with 1% murine GM-CSF and 2.5 ng/ml recombinant murine IL-4 (Cat. No. 12340043, Immunotools). On day 3 and day 5, medium was exchanged to remove nonadherent cells and to supplement cultures with fresh medium containing GM-CSF and mIL-4. Immature DCs were harvested on day 7 of culture. For further treatment 2 × 106 cells/well were seeded into nonadherent six-well plates and cultured in medium containing GM-CSF (1%) and IL-4 (2.5 ng/ml).
8
Murine Splenocyte Isolation and Flow Cytometry
9
Isolation and Culture of Pancreatic Ductal Organoids
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The pancreas from Dkk3 knockout or wildtype mice was minced by subsequent digestion with collagenase/dispase (Roche) for 30 min at 37 °C followed by incubation in accutase (Sigma‐Aldrich) for 30 min at 37 °C. Afterward, cells were filtered through a 40 µm cell strainer (Greiner bio‐one). After centrifugation, cells were resuspended in pancreatic ductal organoid cell (PDC) medium supplemented with 5% growth factor reduced (GFR) Matrigel (Corning, 354 230) and plated onto GFR‐Matrigel coated 12‐well plates. The PDC medium is based on the protocol from Reichert et al.[91 ]
10
Comprehensive Flow Cytometry Tumor Analysis
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For flow cytometry, cells were isolated from spleens, lymph nodes or tumors. Unless indicated, all flow cytometry was done on spleens and tumors from mice when tumors reached a terminal endpoint volume (∼2000 mm3). Prior to staining, tumors were digested using a mix of collagenase (1 mg/mL), DNAse (20 U/mL), and hyaluronidase (100 μg/mL) for 90 minutes at 37°C. Digested tumors, spleens, and LNs were mechanically dissociated by smashing through a 40-µm cell strainer (Greiner Bio-One). Red blood cells were lysed with RBC lysing buffer (Sigma). Fixable Aqua dye (Invitrogen) was added to assess cell viability. Cells were incubated with fluorochrome-conjugated antibodies and fixed with 1% formalin (Sigma). For intracellular staining, a FoxP3 Fix/Perm kit was used according to the manufacturer's instructions (eBioscience). Antibodies used include: PD1 (29F.1A12or RMP1-30), PDL1 (10F.9G2), CD45 (30F11), CD8β (YTS156.7.7), CD4 (GK1.5), NK1.1 (PK136), CD44 (IM7), CD11b (M1/70), Ly6C (HK1.4), F480 (BM8), Ly6G (1A8), FoxP3 (FJK-16S) and/or CTLA4 (UC10-4B9) (all Biolegend). Data were collected using an LSR II flow cytometer (BD Bioscience) and analyzed with FlowJo software (Tree Star).
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