Outgrowth cells derived from EBs were washed and detached from the dish. Cell clumps were removed by passing through a 40 μm cell strainer (Thermo Fisher Scientific). Single outgrowth cells were counted and 3 × 105 cells per pellet were prepared. 3 × 105 outgrowth cells were resuspended in chondrogenic differentiation medium (DMEM, 20% knockout serum replacement, 1 × non-essential amino acids, 1 mM L-glutamine, 1% sodium pyruvate, 1% ITS+ Premix, 10-7M dexamethasone, 50 mM ascorbic acid, 40 μg/mL L-proline supplemented with 50 ng/mL human bone morphogenetic protein 2 and 10 ng/mL human transforming growth factor beta 3) and transferred to a 15 mL conical tube. Cells were centrifuged at 750 × g for 5 minutes. Generated pellets were maintained for 30 days and media was changed every other day. BMSCs were used as a positive control.
40 μm cell strainer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 40-μm cell strainer is a laboratory equipment designed for the filtration and separation of cells or other particulates. It features a 40-micron mesh size membrane that allows the passage of smaller particles while retaining larger ones. This product is commonly used in cell culture and tissue processing workflows to remove debris, clumps, or unwanted materials from cell suspensions.
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Lab products found in correlation
Dnase 1, by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Lsr 2 flow cytometer, by BD (4 mentions)
Hyaluronidase, by Merck Group (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Elisa kit, by BioLegend (2 mentions)
Ova siinfekl, by InvivoGen (2 mentions)
Wt1 rmfpnapyl, by MBL Life Science (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Red blood cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
Dispase 2, by Thermo Fisher Scientific (2 mentions)
Collagenase type 1, by Merck Group (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Renal epithelial cell growth medium, by PromoCell (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
77 protocols using 40 μm cell strainer
1
Chondrogenic Differentiation of Outgrowth Cells
2
Evaluating T Cell Responses to Vaccine Formulations in Mice
3
Isolation and Osteogenic Differentiation of hMSCs from Periodontal Membranes
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hMSCs were isolated from fresh periodontal membranes that were obtained from human teeth following the protocol described in our previous work.46 (link) Briefly, healthy human teeth were obtained from three young individuals (12–16 years old) who were treated for orthodontic reasons at the Stomatology Hospital of Xi'an Jiaotong University. In this study, the periodontal membrane surrounding the root was cut and digested in collagenase type I with concentration of 3 mg/ml (Sigma-Aldrich, USA) at 37 °C for 30 min. Subsequently, cell suspensions were filtered with a 40 μm cell strainer (Thermo Fisher Scientific, USA) and cultured with DMEM (Gibco, USA) containing 15% fetal bovine serum (Gibco, USA) and 100 U/ml penicillin streptomycin (Sigma-Aldrich, USA) in the incubator (5% CO2, 37 °C). Limiting dilution technique was used to gain single cell-derived cultures, then hMSCs were multiple colony-derived after 3–4 passage. hMSCs with the same passage were used in each experiment. Identification of the hMSCs through a flow cytometer was confirmed in our previous study.46 (link) For osteogenic differentiation, the growth media were replaced by the osteogenic media containing 10 mM β-glycerophosphate disodium, 50 μg/ml ascorbic acid, and 100 nM dexamethasone.
4
Fecal Collection and Transplant in Mice
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To collect feces, mice were housed individually in regular cages without any padding for 1-2 h, and feces were collected manually and chilled immediately with ice until use. For each group, 1 g feces was weighed, homogenized with 2.5 mL sterilized phosphate buffered saline using a micro-tube homogenizer, and filtered with a 40 μm cell strainer (Thermo Scientific) to remove any large debris. Prepared fecal homogenates were stored on ice and used within 2 h. For FMT, 200 μL fecal homogenates were given to mice by oral gavage every 3 d.
5
Preparation of Aspergillus Conidial Suspension
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A. fumigatus CBS144-89 (Thau et al., 1994 (link)), CNRMA 15.354 and A. flavus CI1698 (Wong et al., 2021b ) were the clinical isolates used in this study. They were maintained at ambient temperature on 2% malt agar slant for 12–15 days before collecting their conidia using 0.05% Tween-80. The conidial suspension was passed through 40-μm cell strainer (ThermoScientific) to avoid any mycelial contamination.
6
Rat Lung Tissue Digestion and Single Cell Isolation
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Rat lung tissue was minced and digested using collagenase IV (200 U/mL; Thermo Fisher Scientific) and DNAse 1 (200 U/mL; Millipore Sigma) at 37°C for 2 h with regular agitation. Single cell suspensions of digested lung were prepared by passing through a 40 μm cell strainer (Thermo Fisher Scientific) and centrifuging at 400 × g for 5 min. The cell pellet was then resuspended in 3 mL of 1X Red Cell Lysis Solution (Milteny Biotec) and incubated for 2 min at room temperature. The solution stopped by dilution with T-cell media and centrifuged at 400 × g for 5 min. The supernatant was discarded, and the cell pellet resuspended in 10 mL of PBS and counted.
7
Mouse Colon LP Cell Isolation
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LP cells were obtained from mouse colon by enzymatic digestion as described previously (49 (link)). Briefly the large intestines of mice were excised and soaked in ice cold PBS, before removing excess fat and feces, opened longitudinally and washed in Hank's balanced salt solution (HBSS; Sigma) 2% fetal calf serum (FCS; Sigma) before being cut into 0.5 cm sections. A further HBSS containing 2 mM EDTA (Gibco) wash step was used to remove mucous before digestion in complete RPMI (cRPMI) (2 mM L-glutamine{Gibco}, RPMI 1640, 100 μg/ml penicillin, 100 μg/ml streptomycin and 10% FCS, {all Sigma}) containing 0.5 U/ml Liberase TM and 0.1 mg/ml Type IV DNAse from bovine pancreas (both Sigma) for 45 min in a shaking incubator at 180 rpm, 37°C. Cells were then washed in cRPMI, passed through a 40-μM cell strainer (Thermo Fisher Scientific) with the aid of a syringe plunger before resuspension to the required concentration.
8
Isolation of Murine Lung Cell Suspensions
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Lungs were harvested from mice, and single-cell suspensions were prepared as previously described (82 (link)). Briefly, the lungs were thoroughly perfused with cold PBS via the left atrium to remove residual blood in the vasculature. Lung lobes were separated, collected, and digested with dispase II (15 U/ml) (Thermo Fisher Scientific, #17105041) in PBS for 45 min at room temperature and mechanically dissociated by pipetting in sort buffer (DMEM + 2% CC + 1% P/S; referred to as “SB”). Next, cell suspensions were filtered by the 40-μm cell strainer (Thermo Fisher Scientific, #352340) and treated by red blood cell lysis buffer (Thermo Fisher Scientific, A1049201) for 5 min, and the cell suspension was incubated in SB containing 1:1000 deoxyribonuclease I (DNase I) (MilliporeSigma, #D4527) for 45 min at 37°C. Whole lung cell suspensions were then used for subsequent experiments.
9
Immunophenotyping of Lymphoid Cells
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Single cells from thymus, spleen, and lymph nodes were prepared by passing the tissue through a 40 μm cell strainer (Thermo Fisher) using PBS and a syringe plunger. Erythrocytes in blood and spleen were lyzed in lysis buffer (0.16 M NH4Cl, 0.13 M EDTA, and 12 mM NaHCO3), the cells were washed in flow cytometry buffer (2% fetal bovine serum and 2 mM EDTA in PBS) and counted in an automated cell counter (Sysmex). After FcR-blockage (anti-mouse CD16/CD32, BD Biosciences), antibodies specific for the following markers were used: CD4 (GK1.5, Biolegend or RM4-5, BD Biosciences), CD8a (53-6.7, Biolegend), CD44 (IM7, Biolegend), CD25 (PC61, BD Biosciences), CD24 (M1/69, BD Biosciences), and Qa-2 (1-1-2, BD Biosciences). Immunostained cells were analyzed on a FACS Canto II, Accuri C6, or FACS Aria (BD Biosciences). Data were analyzed using FlowJo (Tree Star) and fluorochrome-minus-one staining was used as controls.
10
Fixation, Permeabilization, and Staining of Cells
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1 × 106 live cells were filtered through a 40 μm cell strainer (Thermo Fisher Scientific cat. 22363547) and fixed with 4% PFA for 20 min at RT. Upon fixation, cells were centrifuged at 700 g for 2 min and supernatant was removed. Cells were then permeabilized and stained using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences cat. 554714) as per manufactures instructions. Primary antibodies were incubated for 1 h and secondary antibodies for 30 min according to the dilutions in Additional file 1 : Table S8. Cells were resuspended in fluorescence-activated cell sorting buffer (2% FCS, 2 mM EDTA in DPBS) and kept on ice until flow cytometry acquisition and analysis.
Isotype controls were used to accurately gate positive staining and data were acquired using the CytoFLEX S flow cytometer and analyzed using the CytExpert software (Beckman Coulter).
Isotype controls were used to accurately gate positive staining and data were acquired using the CytoFLEX S flow cytometer and analyzed using the CytExpert software (Beckman Coulter).
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