MDCKII-hMDR1 cells were obtained from Solvo Biotechnology, Hungary. DMEM, fetal bovine serum, Glutamax-100, Antibiotic/Antimycotic, DMSO, Dulbecco’s phosphate-buffered saline, and MEM Non-essential amino acids were purchased from Sigma (St. Louis, MO, USA). Bi-directional permeability and P-glycoprotein substrate assessments were carried out in Madin-Darby canine epithelial cells with the over-expressed human MDR1 gene (MDCKII-MDR1), coding for P-glycoprotein. Experimental procedures, as well as cell culture conditions, were the same as previously described [53 (link)]. Briefly, compounds (10 µM, 1% DMSO v/v) in duplicate were incubated at 37 °C for 60 min with cell monolayer on 24-well Millicell inserts (Millipore, Burlington, MA, USA) with and without the P-glycoprotein inhibitor Elacridar (2 µM, International Laboratory, San Francisco, CA, USA). The inhibition of P-glycoprotein was verified by amprenavir (Moravek Biochemicals Inc, Brea, CA, USA) and monolayer integrity using Lucifer yellow (Sigma, St. Louis, MO, USA). LC-MS/MS measured compound concentrations and Lucifer yellow was measured on an Infinite F500 (Tecan, Männedorf, CH, Switzerland) using excitation of 485 nm and emission of 530 nm.
Millicell insert
Manufactured by Merck Group
168 citations
Sourced in United States, Germany, Italy, United Kingdom, Canada
About the product
Millicell inserts are a type of laboratory equipment used for cell culture applications. They provide a porous membrane that allows for the exchange of media and gases between the upper and lower chambers of a cell culture system. The inserts are designed to be used in multi-well plates, enabling researchers to conduct experiments with different cell types or experimental conditions simultaneously.
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Check compatibility
168 protocols using «millicell insert»
1
Bidirectional Permeability and P-Glycoprotein Substrate Assay
2
Ex vivo Tumor Growth Assay Using Organotypic Slice Cultures
For generating adult organotypic slice cultures (OTC) and perform ex vivo tumor growth assay we followed established procedures by Haydo et al., Linder et al. and Gerstmeier et al. [18 , 26 (link), 63 ]. Briefly, this experiment utilizes OTC, by dissecting mouse brains and generating slices into 150 μm thick slices using a vibratome. These slices were cultured on Millicell inserts (Sigma) in six-well plates with FCS-free medium. Tumor spheres were placed onto the brain slices, and treatments (5 µM CurD and 2 Gy fractioned IR) were administered every three days (Monday, Wednesday and Friday) (Fig. 5 A).
3
Differentiation of A549 Cells at ALI
Membranes of Millicell inserts (0.4-µm pore, 12 mm inserts, polycarbonate, Millipore Sigma, Burlington, MA, USA) were pre-coated with 70 μg of collagen type IV from human placenta (Millipore Sigma, Burlington, MA, USA). Single cell suspensions of A549 cells were seeded on apical surfaces of collagen-pre-coated membranes with a density of 2.5 × 105 cells per well using prepared DMEM described above. The culture medium on the apical side was removed 24 h after the seeding to establish the ALI condition. The ALI cultured cells were refreshed with DMEM in the bottom of the insert at two-day intervals and allowed to differentiate for 14 days (Wu et al. 2017 (link)).
4
Transwell Assay for HUVEC Migration
HUVECs were harvested and suspended in DMEM without FBS. A 400 µL cell suspension containing 10,000 cells was seeded into the upper chamber of a Millicell insert (Millipore, USA). NPC cell-conditioned medium (600 µL) was added to the lower chamber. After 36 h, cells in the Transwell chambers were fixed with 4% formalin (Sevicebio, Wuhan, China) and stained with 0.1% crystal violet for 30 min. Migrated cells were counted post washing with PBS and imaged using a microscope (Olympus) [27 (link)].
Corresponding organizations : Nanjing Drum Tower Hospital, Nanjing Medical University, Xuzhou Medical College
5
Culture and Reconstruction of Mammalian Oocyte Complexes
To prepare TZP-free DOs, groups of 4–14 DOs collected from early antral follicles from at least three biological replicates were cultured individually in 12 µl microdrops of culture medium
covered with paraffin oil in Petri dishes (Falcon No. 351007; BD Biosciences, Franklin Lakes, NJ, USA) at 38.5°C under a controlled humidified atmosphere of 5% O2, 5%
CO2, and 90% N2 for 24 h as described previously [21 (link)]. The basic culture medium was α-minimum essential medium (αMEM, Cat. No.
11900-024; Invitrogen, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS; ICN Biomedicals, Aurora, OH, USA), 50 µg/ml ascorbic acid 2-glucoside (Hayashibara Biochemical
Laboratories, Okayama, Japan), 55 µg/ml cysteine, 0.05 µM dexamethasone, 4 mM hypoxanthine, 4% (w/v) polyvinylpyrrolidone (molecular weight 360,000), 2.2 mg/ml sodium bicarbonate, and 0.08
mg/ml kanamycin sulfate [24 (link)]. Based on a previous report [23 (link)], the culture medium was supplemented with 10 ng/ml
17β-estradiol and 10 ng/ml androstenedione (Tokyo Chemical Industry, Tokyo, Japan).
To reconstruct oocyte-granulosa cell complexes and oocyte-mural granulosa cell complexes, 2–3 masses of GCs from secondary follicles and masses of MGCs, larger than those of DOs, from early
and late antral follicles were prepared as described above. Collected GCs and MGCs were cocultured with TZP-free DOs individually in 12 µl microdrops of the culture medium in Petri dishes
(Falcon No. 351007) for 24 h. After coculture, the reconstructed complexes in which GCs and MGCs adhered to DOs (DO+GCs and DO+MGCs) were transferred to Millicell inserts (cell culture
inserts 0.4 µm, 30 mm diameter; Merck Millipore, Darmstadt, Germany) in Petri dishes (Falcon No. 351008) and cultured for 4 or 12 days. The reconstructed complexes were cultured at 38.5°C
under a controlled humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 5 days, followed by culture in an atmosphere of 5% CO2 in the air for 7
days [25 (link)]. The day on which the DOs were collected was designated as day 0, and half of the culture medium was replaced with fresh medium every other
day after day 6.
covered with paraffin oil in Petri dishes (Falcon No. 351007; BD Biosciences, Franklin Lakes, NJ, USA) at 38.5°C under a controlled humidified atmosphere of 5% O2, 5%
CO2, and 90% N2 for 24 h as described previously [21 (link)]. The basic culture medium was α-minimum essential medium (αMEM, Cat. No.
11900-024; Invitrogen, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS; ICN Biomedicals, Aurora, OH, USA), 50 µg/ml ascorbic acid 2-glucoside (Hayashibara Biochemical
Laboratories, Okayama, Japan), 55 µg/ml cysteine, 0.05 µM dexamethasone, 4 mM hypoxanthine, 4% (w/v) polyvinylpyrrolidone (molecular weight 360,000), 2.2 mg/ml sodium bicarbonate, and 0.08
mg/ml kanamycin sulfate [24 (link)]. Based on a previous report [23 (link)], the culture medium was supplemented with 10 ng/ml
17β-estradiol and 10 ng/ml androstenedione (Tokyo Chemical Industry, Tokyo, Japan).
To reconstruct oocyte-granulosa cell complexes and oocyte-mural granulosa cell complexes, 2–3 masses of GCs from secondary follicles and masses of MGCs, larger than those of DOs, from early
and late antral follicles were prepared as described above. Collected GCs and MGCs were cocultured with TZP-free DOs individually in 12 µl microdrops of the culture medium in Petri dishes
(Falcon No. 351007) for 24 h. After coculture, the reconstructed complexes in which GCs and MGCs adhered to DOs (DO+GCs and DO+MGCs) were transferred to Millicell inserts (cell culture
inserts 0.4 µm, 30 mm diameter; Merck Millipore, Darmstadt, Germany) in Petri dishes (Falcon No. 351008) and cultured for 4 or 12 days. The reconstructed complexes were cultured at 38.5°C
under a controlled humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 5 days, followed by culture in an atmosphere of 5% CO2 in the air for 7
days [25 (link)]. The day on which the DOs were collected was designated as day 0, and half of the culture medium was replaced with fresh medium every other
day after day 6.
Top 5 most cited protocols using «millicell insert»
1
Isolation and Characterization of Human Alveolar Epithelial and Macrophage Cells
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Corresponding organizations : National Jewish Health, University of Colorado Denver, Boston University
2
HBoV1 Infection in Airway Epithelial Cells
Fully differentiated primary B- (each of the three distinct subtypes), CuFi- and NuLi-HAE were cultured in Millicell inserts (0.6 cm2; Millipore) and inoculated with 150 µl of purified HBoV1 (1×107 gc/µl in phosphate buffered saline, pH7.4; PBS) from the apical surface (at a multiplicity of infection, MOI, of ∼750 gc/cell; an average of 2×106 cells per insert). For each of the HAE, a 2-h incubation was followed by aspiration of the virus from the apical chamber and by three washes of the cells with 200 µl of PBS to remove unbound virus. The HAEs were then further cultured at an ALI.
For conventional monolayer cells, cells cultured in chamber slides (Lab-Tek II; Nalge Nunc) were infected with purified HBoV1 at an MOI of 1,000 gc/cell.
For conventional monolayer cells, cells cultured in chamber slides (Lab-Tek II; Nalge Nunc) were infected with purified HBoV1 at an MOI of 1,000 gc/cell.
Corresponding organizations : University of Kansas Medical Center, Central China Normal University, University of Iowa, University of Kansas, University of Helsinki
3
SARS-CoV-2 Infection of Respiratory Epithelial Cells
Nasal cells were expanded using the conditionally reprogrammed cell (CRC) method (Gentzsch et al., 2017 (link)) or in Pneumacult EX Plus media (Stem Cell Technologies) (Speen et al., 2019 (link)) and then cultured on porous Transwell (Corning) supports in Pneumacult air liquid interface (ALI) media (Stem Cell Technologies). Human LAE and SAE cells were cultured as previously described (Fulcher and Randell, 2013 (link), Okuda et al., 2019 (link)). Briefly, Isolated LAE and SAE cells were co-cultured with mitomycin-treated 3T3 J2 cells on collagen-coated tissue culture plastic dishes in DMEM media supplemented with 10 μM Y-27632 (Enzo Life Science). At 70%–90% confluence, LAE and SAE cells were passaged and sub-cultured for expansion. P2 LAE and SAE cells were transferred to human placental type IV collagen-coated, 0.4 μm pore size Millicell inserts (Millipore, PICM01250). The LAE and SAE cells were seeded at a density of 2.8 × 105 cells/cm2 and cultured in UNC ALI media. Upon confluence, cells were maintained at an ALI by removing apical media and providing UNC ALI media to the basal compartment only. Medium was replaced in the basal compartment twice a week, and the apical surfaces were washed with PBS once a week. After 28 days, LAE and SAE cells were utilized for SARS-CoV-2 recombinant viruses infection. Human type II pneumocytes (AT2) were prepared and cultured on porous supports as previously described (Bove et al., 2010 (link)). The AT2 cells are grown in DMEM with P/S and 10% FBS and switched to 4% FBS 24 h prior to infection. Cells were studied within three days and after five days, as they transdifferentiate into type I pneumocyte (AT1)-like cells. For serum-free and feeder-free AT2 cell cultures (mixed AT1/AT2 culture), human lung pieces (∼2 gm) were washed twice with PBS containing 1% Antibiotic-Antimycotic and cut into small pieces. Visible small airways and blood vessels were carefully removed to avoid clogging. Then samples were digested with 30 mL of enzyme mixture (collagenase type I: 1.68 mg/mL, dispase: 5U/mL, DNase: 10 U/mL) at 37°C for 45 min with rotation. The cells were filtered through a 100 μm strainer and rinsed with 15 mL PBS through the strainer. The supernatant was removed after centrifugation at 450x g for 10 min and the cell pellet was resuspended in red blood cell lysis buffer for five minutes, washed with DMEM/F12 containing 10% FBS and filtered through a 40 μm strainer. To purify human AT2 cells, approximately two million total lung cells were resuspended in SF medium and incubated with Human TruStain FcX (BioLegend) followed by incubation with HTII-280 antibody (Terrace Biotech). The cells were washed with PBS and then incubated with anti-mouse IgM microbeads. The cells were loaded into LS column (Miltenyi Biotec) and labeled cells collected magnetically. HTII-280+ human AT2 cells (1-3 × 103) were resuspended in culture medium. Serum-free feeder free medium and AT2 differentiation medium will be described elsewhere (S.V. and PRT et al., currently under revision in Cell Stem Cell). Culture plates were coated with Cultrex reduced growth factor basement membrane extract, Type R1 and cultured for five days followed by changing medium to AT2 differentiation medium for additional five days.
Human primary lung microvascular endothelial cells (MVE) and fibroblasts (FB) were grown as previously described (Scobey et al., 2013 (link)). For MVE cells, peripheral lung tissue minus the pleura was minced, digested with dispase/elastase, and cells were grown in EGM-2 media plus FBS (Lonza). Two or three rounds of CD31 bead purification (Dynabeads; Life Technologies) resulted in > 95% CD31-positive cells by flow cytometry that were used between passages 5 and 10. FBs were obtained by finely mincing distal human lung tissue and plating on scratched type 1/3 collagen-coated dishes in Dulbecco’s modified Eagle medium with high glucose (DMEMH) media plus 10% FBS, antibiotics, and antimycotics. Cells were released using trypsin/EDTA and subcultured in DMEMH, 10% FBS and P/S. The subcultured cells were elongated, spindly and negative for CD31 and pan-cytokeratin by flow cytometry and immunofluorescence, respectively.
icSARS-CoV-2-GFP virus infections were performed using well differentiated air-liquid interface (ALI) cultures of five donor specimens of human nasal epithelial (HNE) and large airway epithelial (LAE) cells using an MOI of three. Small airway epithelial (SAE) cell ALI cultures were created as previously described (Okuda et al., 2019 (link)). Paired LAE / SAE cells were inoculated with a SARS-CoV-2 clinical isolate, icSARS-CoV-2-WT, and icSARS-CoV-2-GFP, as well as wild-type icSARS-CoV-Urbani and icSARS-CoV-GFP on LAE, using an MOI of 0.5 for each virus. Transwell-cultured primary cells were inoculated with 200ul of virus via the apical surface and allowed to incubate at 37°C for two h. Following incubation, virus was removed, and cells were washed twice with 500ul PBS. Cells were returned to 37°C for the remainder of the experiment and observed for fluorescent signal, when appropriate, every 12-24 h. 100ul PBS was added to the apical surface of each culture and allowed to incubate for 10 min at 37°C in order to obtain an apical wash sample, at time points for analysis of viral replication by plaque assay. At the last time point, cells were lysed with 500ul TRIzol reagent (Invitrogen) to obtain total final RNA for analysis.
Human primary lung microvascular endothelial cells (MVE) and fibroblasts (FB) were grown as previously described (Scobey et al., 2013 (link)). For MVE cells, peripheral lung tissue minus the pleura was minced, digested with dispase/elastase, and cells were grown in EGM-2 media plus FBS (Lonza). Two or three rounds of CD31 bead purification (Dynabeads; Life Technologies) resulted in > 95% CD31-positive cells by flow cytometry that were used between passages 5 and 10. FBs were obtained by finely mincing distal human lung tissue and plating on scratched type 1/3 collagen-coated dishes in Dulbecco’s modified Eagle medium with high glucose (DMEMH) media plus 10% FBS, antibiotics, and antimycotics. Cells were released using trypsin/EDTA and subcultured in DMEMH, 10% FBS and P/S. The subcultured cells were elongated, spindly and negative for CD31 and pan-cytokeratin by flow cytometry and immunofluorescence, respectively.
icSARS-CoV-2-GFP virus infections were performed using well differentiated air-liquid interface (ALI) cultures of five donor specimens of human nasal epithelial (HNE) and large airway epithelial (LAE) cells using an MOI of three. Small airway epithelial (SAE) cell ALI cultures were created as previously described (Okuda et al., 2019 (link)). Paired LAE / SAE cells were inoculated with a SARS-CoV-2 clinical isolate, icSARS-CoV-2-WT, and icSARS-CoV-2-GFP, as well as wild-type icSARS-CoV-Urbani and icSARS-CoV-GFP on LAE, using an MOI of 0.5 for each virus. Transwell-cultured primary cells were inoculated with 200ul of virus via the apical surface and allowed to incubate at 37°C for two h. Following incubation, virus was removed, and cells were washed twice with 500ul PBS. Cells were returned to 37°C for the remainder of the experiment and observed for fluorescent signal, when appropriate, every 12-24 h. 100ul PBS was added to the apical surface of each culture and allowed to incubate for 10 min at 37°C in order to obtain an apical wash sample, at time points for analysis of viral replication by plaque assay. At the last time point, cells were lysed with 500ul TRIzol reagent (Invitrogen) to obtain total final RNA for analysis.
Corresponding organizations : University of North Carolina at Chapel Hill, Lung Institute, National Heart Lung and Blood Institute, National Institutes of Health, Environmental Protection Agency, Case Western Reserve University, Shantou University Medical College, Dalhousie University, Shantou University, University of New Mexico, Cornell University, Duke Medical Center
4
Bioluminescence Monitoring of Lung Slice Circadian Rhythms
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Corresponding organizations : University of Manchester, Wythenshawe Hospital, GlaxoSmithKline (United Kingdom)
5
Neonatal Rat Hippocampal Slice Culture
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Corresponding organizations : Augusta University, University of Montana, Augusta University Health
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