Femurs from pigs between 80 and 100 kg were cleared of all muscle and sinew. Bone was then washed with 70% ethanol. A sterilized junior hacksaw was used to cut transversely across bone to expose bone marrow under sterile conditions. A total of 5 g of bone marrow per 50-ml tube was suspended in 40 ml of complete medium and centrifuged at 600 × g for 8 min to remove fat. Red blood cells were lysed via incubation with ammonium-chloride-potassium lysis buffer (A1049201; Gibco) for 3 min. Cells were washed in 10 ml of complete medium and passed through a 70-mm cell strainer (2236348; Fisherbrand) prior to centrifugation. Cell pellet was dislodged before plating on 20-cm petri dishes (Sarstedt) in 25 ml of complete medium (DMEM, high glucose, GlutaMAX, supplemented with 10% FCS and 1% penicillin-streptomycin) and 5 ml of syringe-filtered L929 supernatant (a source of macrophage colony-stimulating factor [M-CSF]). Cells were cultured for 6 days before assessment of purity by flow cytometry.
A1049201
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The A1049201 is a laboratory centrifuge produced by Thermo Fisher Scientific. It is designed for general-purpose applications that require centrifugation of samples. The product specifications and technical details are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sh30910, by Thermo Fisher Scientific (1 mentions)
Ab102884, by Abcam (1 mentions)
Ab102918, by Abcam (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Bambanker, by Nippon Genetics (1 mentions)
Collagenase 4, by Worthington (1 mentions)
Collagenase 2, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Fortessa x20, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
17 protocols using a1049201
1
Isolation of Bone Marrow-Derived Macrophages
2
Tumor Dissociation for Single-Cell Analysis
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Patient tumors were processed as described previously (66 (link)). Adjustments are described: tumors were obtained on the day of Mohs micrographic surgery and washed in cold DMEM [Gibco 11995-065] supplemented with 10% FBS [Thermo Scientific SH30910.03]. Any fat was removed from tumor samples using a scalpel. Then, tumors were then cut into small pieces using a razor and resuspended in 10 mL of DMEM/10% FBS, 10 mg/mL collagenase II [Sigma C2674] and 10 U DNase I [Sigma 00453869] for 20 min at 37°C. After incubation, the suspension was vortexed 1× for 30 s followed by pipetting sequentially through 25, 10 and 5 ml pipettes for 1 min each. Next, the cell suspension was filtered through a 70-μm filter [Fisher 22363548] and spun at 300 g for 5 min. After spinning, the supernatant was removed, and ACK red blood cell lysis was performed according to the manufacturer's protocol as needed [Gibco a10492-01].
3
Multicolor Phenotyping of Pig Lung Cells
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A total of 100 μg of lung tissue was homogenized in 1 ml of sterile PBS and filtered through a 70-μm cell strainer (2236348; Fisherbrand). Cells were centrifuged and red blood cells lysed using ammonium chloride-potassium lysis buffer (A1049201; Gibco) for 3 min at room temperature and washed with 1 ml of PBS prior to staining with the following mouse anti-pig antibodies: CD11R3 (MCA2309), CD163 (clone 2A10), SLA class II, and granulocyte antibody (clone 6D10) (AbD Serotech). Each purified anti-pig antibody was labeled with a fluorophore using Abcam phycoerythrin (PE; ab102918), allophycocyanin (APC)-Cy5.5 (ab102855), fluorescein isothiocyanate (FITC; ab102884), and rhodamine (ab188286) conjugation kits.
4
Single-cell dissociation of resected glioma
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Resected glioma tissue samples were immediately placed on ice and transferred to the laboratory for single cell dissociation within 2–3 hr after resection. Human brain tissue was manually minced using razor blades and enzymatically dissociated at 37 °C for 30 min with 1 mg/ml collagenase-4 (#LS004188, Worthington Biochemical Corporation, USA) and 250 U/ml DNAse1 (#10104159001, Roche, Switzerland) in a buffer containing Hank’s Balanced Salt Solution (HBSS) with Ca2+/Mg2+, 1% MEM non-essential amino acids (Gibco, USA), 1 mM sodium pyruvate (Gibco), 44 mM sodium bi-carbonate (Gibco), 25 mM HEPES (Gibco), 1% GlutaMAX (Gibco) and 1% antibiotic-antimycotic (Sigma-Aldrich, USA). Cells were filtered and separated from dead cells, debris and myelin by a 0.9 M sucrose (#84100, Sigma-Aldrich) density gradient centrifugation. Upon ACK-lysis for removal of erythrocytes (#A1049201, Gibco) the now generated single-cell suspension (SCS) was washed, counted and frozen in Bambanker (#BB01, Nippon Genetics, Germany) in liquid nitrogen until use.
5
Isolation of Tumor-Infiltrating Cells
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Patient tumors were obtained on the day of surgery and washed in cold 1× PBS. Any fat was removed from tumor samples using a scalpel. Then, tumors were then cut into small pieces using a razor and resuspended in 10 mL of RPMI [Gibco 11875-093] supplemented with 5% human serum [Sigma H4522-100], 5 mg gentamicin [Sigma H0887], 5 mg/mL collagenase II [Sigma C2674] and 10 U DNase I [Sigma 00453869] for 10 min at 37 °C. After incubation, the suspension was vortexed 1× for 30 s followed by pipetting sequentially through 25, 10, and 5 mL pipettes for 1 min each. Next, the cell suspension was filtered through a 70-μm filter [Fisher 22363548] and spun at 450 g for 10 min. After spinning, the supernatant was removed, and ACK red blood cell lysis was performed according to the manufacturer’s protocol as needed [Gibco a10492-01]. Cells were then stained as described below prior to cell sorting and FACS analysis.
6
Tumor Dissociation for Single-Cell Analysis
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Patient tumors were processed as described previously 60 (link) . Adjustments are described: tumors were obtained on the day of Mohs micrographic surgery and washed in cold DMEM [Gibco 11995-065] supplemented with 10% FBS [Thermo Scientific SH30910.03]. Any fat was removed from tumor samples using a scalpel. Then, tumors were then cut into small pieces using a razor and resuspended in 10 mL of DMEM/10% FBS, 10 mg/mL collagenase II [Sigma C2674] and 10 U DNase I [Sigma 00453869] for 20 min at 37 °C. After incubation, the suspension was vortexed 1× for 30 s followed by pipetting sequentially through 25, 10, and 5 mL pipettes for 1 min each. Next, the cell suspension was filtered through a 70-μm filter [Fisher 22363548] and spun at 300 g for 5 min. After spinning, the supernatant was removed, and ACK red blood cell lysis was performed according to the manufacturer's protocol as needed [Gibco a10492-01].
7
Evaluating Axitinib Effects on Hematopoietic Stem Cells
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Fully engrafted animals administered with axitinib (or vehicle control) and three cycles of DSS were harvested on day 70. Peripheral blood was collected by a submandibular puncture. BM was isolated from long bones by spin-flush method. Red blood cells were depleted by incubating with ammonium-chloride-potassium lysis buffer (A1049201; Thermo Fisher Scientific) on ice for 15 min. Analysis of the donor-derived (CD45.2+) stem and progenitor cell populations was performed on single-cell suspensions by flow cytometry. LT-HSCs were defined as Lineage−Sca1+c-Kit+ (LSK) CD150+CD48−, granulocyte/macrophage progenitors–LSK CD16/32+CD34+, using the antibody cocktail in Table 3 .
8
Neutrophil Chemotaxis Transwell Assay
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Neutrophils were derived from whole blood from healthy donors following a red cell lysis step (Thermofisher A1049201) then resuspended at a concentration of 107 cells/mL in DMEM; 200 mL was applied to the upper chamber of a 5-µm transwell (24-well format), 6.5 mm membrane. Diluted Th17 supernatant (500 µL) was placed in the lower transwell chamber to act as chemo-attractant. Co-cultures were incubated for 5 hours at 37°C in a 5% CO2 culture incubator and migrated neutrophils (CD18 FITC) were enumerated using flow cytometry (BD Fortessa X20). Further details on the chemotaxis assay are provided in the online supplementary information .
9
Murine Peripheral Blood Immune Cell Profiling
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100 µl of blood was collected in EDTA‐coated tubes and red blood cells were removed by incubation (3×) in Ammonium‐Chloride‐Potassium (ACK, Thermo Fisher A1049201) solution for 5 min at room temperature. Peripheral blood mononuclear cells were then resuspended in FACS buffers (2% BSA in PBS) and incubated for 20 min on ice with the following antibodies: anti‐CD45‐Pac. Blue (clone 30‐F11), CD3e‐PE (Clone 145‐2C11), CD11b‐APC (Clone M1/70), CD19‐BV650 (Clone 6D5), Ly6C‐PE/Dazzle (Clone HK1.4), and Ly6G‐PerCP Cy5.5 (1A8) in 1:100 dilution ratio. Stained samples were washed once and resuspended in FACS buffer containing DAPI (4 µg/ml). Flow cytometry was performed on a BD Fortessa using FACSDiva software with single stain compensation controls acquired on the same day. Data were analyzed using FlowJo analysis software (FlowJo, LLC). All antibodies were purchased from Biolegend (San Diego, CA) and used at the manufacturer’s recommended concentrations.
10
Isolation and Characterization of Mouse Bone Marrow Mesenchymal Stem Cells
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Bone marrow (BM) cells were flushed out from tibiae and femurs of 6‐week‐old or 8‐week‐old mice with phosphate buffered saline (PBS). Red blood cells were lysed using lysis buffer (ThermoFisher Scientific A1049201, Waltham, MA USA). (a) For CFU‐f and ALP positive CFU‐f (CFU‐fAP) colony formation assays, BM cells were cultured in 60 mm dishes at 4 × 106 cells per dish in α‐minimum Eagle's medium (MEM) containing 10% fetal calf serum (FCS) (Hyclone Laboratories, South Logan, UT, USA) with or without 50 μg/ml ascorbic acid and 10 mM β‐glycerophosphate for 12 days. At the end of the culture period, cells were stained for CFU‐f or CFU‐fAP. (b) For BM‐MSCs cultures, BM‐MSCs are a heterogeneous population of postnatal precursor cells with the ability to adhere to culture dishes, producing CFU‐f 23 . BM cells were cultured at 4 × 106 cells per dish in 60 mm dishes with α‐MEM containing 10% FCS for 7 days. All the cells were digested, collected, and transferred into a Petri dish as the first passage and continually cultured for another 7 days to allow cell growth to confluence. The third generation BM‐MSCs were analyzed by Western blot for Bmi1 expression.
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