Cryopreserved, primary normal human bronchial epithelial (NHBE) cells from one donor were purchased from Lonza (CC2540, lot 307177, 40Y Female; Walkersville, MD). Donor had no history of smoking or preexisting lung conditions. The subjects cause of death and presence of nonlung-related diseases remains unknown. Cryopreserved cell stock was expanded in bronchial epithelial growth medium (BEGM, Lonza) at a density of 3500 cells/cm2. Cells were frozen down at passage 2, expanded, and seeded onto human collagen-IV (Sigma, St. Louis, MO) coated 6.5 mm Transwell inserts (Costar, Corning, NY) at a density of 3.0 × 105 cells/cm². Cultures were maintained for 3 days in immersed conditions until confluency was reached. An air–liquid interface (ALI) was established by removing the apical medium and incubating with differentiation medium in the basolateral compartment only. Differentiation medium was comprised of a 50:50 mixture of LHC-basal medium (Gibco, Carlsbad, CA) and DMEM-H (Gibco) and supplemented as previously described by Fulcher et al. (2005 (link)). Cells were maintained at an air–liquid interface for up to 3 weeks, with differentiation media replenished every other day.
Human collagen 4
Manufactured by Merck Group
Sourced in United States
Human collagen IV is a laboratory product that serves as a key structural component of the extracellular matrix. It is a glycoprotein that provides support and cohesion to cells within various tissues.
Automatically generated - may contain errors
Lab products found in correlation
Human fibronectin, by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Human collagen 1, by Merck Group (3 mentions)
Purecol, by Advanced BioMatrix (2 mentions)
Laminin, by Merck Group (2 mentions)
Pna fitc, by Vector Laboratories (2 mentions)
Gsl 2 fitc, by Vector Laboratories (2 mentions)
Fitc lel, by Vector Laboratories (2 mentions)
Rca 1 fitc, by Vector Laboratories (2 mentions)
Ecl fitc, by Vector Laboratories (2 mentions)
Mal 2 fitc, by Vector Laboratories (2 mentions)
Murine laminin, by Merck Group (2 mentions)
Collagen 4, by Merck Group (2 mentions)
Transwell, by Corning (2 mentions)
Transwell insert, by Corning (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Evom2 epithelial voltohmmeter, by World Precision Instruments (2 mentions)
Cc 2540, by Lonza (1 mentions)
23 protocols using human collagen 4
1
Differentiation of Primary Human Bronchial Epithelial Cells
2
Colonoid Infection Assay with EAEC
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Human colonoid line 75C was established from de-identified biopsy tissue from a healthy subject provided informed consent at Johns Hopkins University. All methods were carried out in accordance with guidelines and regulations approved by the Johns Hopkins University Institutional Review Board (IRB# NA_00038329). Colonoids were maintained as previously described [28 (link)]. Colonoid monolayers were cultured in a 96-well plate pre-coated with human collagen IV (30 μg/ml; Sigma-Aldrich, USA) until confluency and then differentiated for 3–4 days. EAEC strains were grown for 16–18 hours without shaking in DMEM-high glucose (Gibco). Approximately 1 x 106 CFUs were added to each well. After a 3 hour incubation, the monolayers were washed three times with PBS, lysed with 1% Triton X-100/PBS, and adherent bacteria were enumerated by dilution and plating on Luria agar.
3
Enteroid-based Infection and Transfection
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Crypt isolation and enteroid propagation were described previously.17 (link) One jejunal enteroid line of blood group A genotype was used to evaluate the bacterial infection parameters and to generate the shRNA KD lines. Three jejunal lines were evaluated in synthetic ST experiments to establish effects were generalizable; results from one line are shown. One jejunal and one colonoid line was used in the comparative study with T84 and Caco-2. The expansion medium (EM) and differentiation medium (DM) have been detailed previously.20 (link) HEMs were established using a published procedure.20 (link) Transwells (catalog number 3470, Corning Life Sciences, Tewksbury, Massachusetts) were coated with human collagen IV (Sigma) at 10 µg/cm2, 4°C overnight, then were washed with ADF, plated with isolated enteroid fragments, and fed with Undifferentiated Monolayer medium (UDM, consists of EM without A83-01 or SB202190) until confluent. The medium was then replaced with DM for 5 d prior to experiments. Transepithelial electrical resistance measurements (EVOM2, World Precision Instruments, Sarasota, Florida) were used to monitor confluency.
4
Airway Epithelial Cell Culture Optimization
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The chip top channel was emptied and subsequently completely filled with 300 μg/mL human collagen IV (Sigma) solution in PBS (approximately 35 μl). Chips were placed in petri dishes containing a small, open container of PBS to promote humidity and kept overnight in the incubator at 37 °C/5%CO2. For optimization experiments, also other coating components and their mixtures were used: 0.1% BSA (Gibco), 30 μg/mL bovine type-1 collagen (PureCol®, Advanced BioMatrix), or human fibronectin (Sigma). We found that BSA and collagen IV coating both resulted in reliable cell adhesion; however, collagen IV coated chips exhibited significantly better viability post seeding and significantly higher ciliation compared to BSA coated chips at day 14 ALI (Supplementary Fig. 6 ).
5
Establishment of Colonoid Monolayers
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Colonoid monolayers were established from colonoid cultures harvested after 7 days. To establish a monolayer, approximately 200 colonoids were completely disaggregated with the help of a 27‐gauge needle. Approximately 100 μl of cell suspension in IntestiCult™ Organoid Growth medium (Human) (Stemcell™ Technologies) with 10 μM Y‐27632 and 100 μg/ml Primocin (InvivoGen) were seeded on permeable polyester filter insert (Corning Transwell, pore size 0.4 μM) coated with 10 μg/ml human collagen IV (Sigma‐Aldrich) solution, in 24‐well plates.27 An additional 50 and 600 μl of IntestiCult™ Organoid Growth medium with Y‐27632 and Primocin was added to the Transwell filter and the well, respectively, and kept in culture for 3 days. Then, expansion medium was substituted with differentiation medium, where 50% v/v IntestiCult (Component B) was substituted by DMEM/Ham's F‐12. The differentiation medium was replaced with fresh differentiation medium after 2 days and colonoid monolayers were then kept in culture for one additional day to reach 3 days of differentiation.
6
Isolation and Purification of Human Cardiac Cells
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Cells were isolated from post-surgically discarded samples of human right atrial appendage and right ventricle myocardium as follows: the tissue was mechanically ground into pieces (1–3 mm3) and then enzymatically treated in 0.1% collagenase NB solution (Life Technologies, USA) at 37 °C for 30 min. After that, the resulting mixture of cells and debris was centrifuged at 300g for 5 min and seeded in plastic dishes coated with human collagen IV (Sigma, USA) in culture media specific for EC or SMC–EGM-2 (Endothelial Cell Growth Medium-2) or SmGM-2 (Smooth Muscle Growth Medium-2) (both—Lonza, Switzerland). Cell culture was maintained in 5% CO2 at 37 °C with 1:2–1:3 passaging using TrypLE Express enzyme (Life Technologies, Denmark). The medium was replaced completely every other day; half of the culture medium was replaced daily. When primary cell culture in EGM-2 reached a monolayer, 106 cells were sorted using magnetic MicroBeads (130-091-935, Miltenyi Biotec, Germany) conjugated with antibodies against human CD31. The procedure of magnetic-activated cell sorting (MACS) was conducted according to the manufacturer’s instructions.
7
HUVEC Cell Migration Assay
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Cell migration was measured using an in vitro wound healing assay. Briefly, HUVEC (28,000 cells/side) were seeded on each side of a culture insert for live cell analysis (Ibidi, Munich, Germany). Inserts were placed in wells of a pre-coated 24-well plate (20 μg/ml human collagen IV and 10 μg/ml human plasma fibronectin (Sigma Aldrich) for 2 h at 37°C) and incubated at 37°C and 5% CO2 to allow cells to grow to confluence. The inserts were then removed with sterile tweezers to create a cell-free area of approximately 500 μm and the cells were treated with NT4 peptide (10 μM) in complete medium. The cells were allowed to migrate in an appropriate incubator. At time point zero and after 24 h, the wound area was observed under an inverted microscope (Zeiss Axiovert 200 microscopy) at 5x magnification and photographed with a Nikon ACT-1 Version 2.63 camera. The percentage of void area with respect to time 0 was determined using ImageJ software, Wound healing Tool Option, after 24 h, when control wells had completely filled the gap.
8
Culturing hPBEC on Collagen IV Coated Inserts
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Clear polyester 6.5 mm cell culture inserts with 0.4 μm pore size (Corning Costar, Cambridge, USA) were pre-coated with 300 μg/mL human collagen IV (Sigma) solution in PBS. hPBEC were seeded at high density (70.000 cells/well) and cultured as above.
9
Integrin-Mediated Cell Adhesion Assay
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Antibody against B4GALT1 was obtained from Abnova (Cat#PAB20512), while that against integrin β1 (CD29) was purchased from BD Transduction Laboratories (Cat#610468). Further, antibody against integrin α 6 was obtained from Cell Signaling Technology Inc. (Cat#3750), while that against GAPDH was purchased from Meridian Life Science. (Cat#H86504M). Functional blocking antibodies for integrin β1 (P4C10) were obtained from Merck Millipore (Cat#MAB1987Z). Further, functional blocking antibodies for integrin α 6 (CD49f) were purchased from Invitrogen (Cat#12-0495-82), and human collagen I, human collagen IV, human fibronectin, laminin, or bovine serum albumin (BSA) were purchased from Sigma Aldrich (St Louis, MO, USA). GSL-II-FITC, LEL-FITC, RCA-I-FITC, ECL-FITC, MAL-II-FITC, and PNA-FITC were purchased from Vector Laboratories (Burlingame, CA, USA).
10
Isolation and Culture of Skin, Gut, and Immune Cells
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Whole-skin punch biopsies and cervix samples were incubated in 5U dispase (Life Technologies) overnight at 4°C followed by manual separation of epidermis and cervical epithelium from dermis or cervical submuocsa respectively followed by 90 min incubation in collagenase III (3 mg/ml; Worthington) with DNase (5 μg/ml; Roche) in RPMI 1640. Epidermal cell suspension was prepared by repeated pipetting. Dermis and submucosa were further processed by Medicon tissue disruptor (BD Biosciences) as previously described (Cheuk et al., 2014 (link)). Ileum biopsies were digested in collagenase II (0.25 mg/ml; Sigma-Aldrich) with DNase (0.2 mg/ml; Roche) in IMDM (Life Technologies) for 30–45 min. Complete RPMI medium was added and the cell suspension were subsequently passed through a 40 μm (gut) / 70 μm (skin or cervix) cell strainer (BD Bioscience). Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll (GE Healthcare) density separation.
P815 cells were purchased from ATCC and maintained in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum [FBS], L-glutamine; all Hyclone). Recombinant IL-15, IL-1β, IL-2, IL-6, IL-7, IL-23, IL-12 and IFN-α (all R&D Systems) were stored at −80°C. Human collagen IV, phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma.
P815 cells were purchased from ATCC and maintained in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum [FBS], L-glutamine; all Hyclone). Recombinant IL-15, IL-1β, IL-2, IL-6, IL-7, IL-23, IL-12 and IFN-α (all R&D Systems) were stored at −80°C. Human collagen IV, phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma.
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