An In Vitro Flow Investigation of the Effect of Stromal Cells on Leukocyte Recruitment

Published: March 29, 2024

Abstract

Source: Munir, H. et al., Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow. J. Vis. Exp. (2015)

The video demonstrates a method for investigating the impact of stromal cells on leukocyte recruitment. In a parallel plate flow chamber model, pro-inflammatory cytokines induce the expression of adhesion receptors by endothelial cells, facilitating neutrophil recruitment and migration. The cross-talk between stromal cells and endothelial cells diminishes neutrophil recruitment to the endothelial cells.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Isolation and Culture of Primary Human Endothelial Cells and Mesenchymal Stem Cells Isolation and culture of human umbilical vein endothelial cells (HUVEC): Put the umbilical cord on a tray with paper towels and spray it with 70% ethanol. Place in a tissue culture hood. Identify the vein and cannulate at both ends. Pl…

Representative Results

Figure 1. Establishing EC-MSC co-culture and analyzing neutrophil recruitment using a flow-based adhesion assay. (A) Micrograph of (i) primary EC and (ii) passage 3 BMMSC grown on tissue culture flasks. (B) Micrograph of EC and MSC cultured on 6-well Transwell filter inserts. (C) …

Divulgations

The authors have nothing to disclose.

Materials

Collagenase Type Ia Sigma C2674 Dilute in 10ml PBS to get a final concentration of 10mg/ml. Store at -20°C in 1ml aliquots.
Dulbecco's PBS Sigma D8662 With calcium and magnesium chloride. Keep sterile and store at room temperature.
1X Medium M199 Gibco 31150-022 Warm in 37 °C water bath before use.
Gentamicin sulphate Sigma G1397 Store at 4°C. Add to M199 500ml bottle.
Human epidermal growth factor Sigma E9644 Store at -20°C in 10µl aliquots.
Fetal calf serum (FCS) Sigma F9665 FCS must be batch tested to ensure the growth and viability of isolated EC. Heat inactivate at 56°C. Store in 10ml aliquots at -20°C.
Amphotericin B Gibco 15290-026 Potent and becomes toxic within a week so fresh complete HUVEC medium must be made up every week. Store at -20°C in 1ml aliquots.
Hydrocortisone Sigma H0135 Stock is in ethanol. Store at -20°C in 10µl aliquots.
Collagenase Type II Sigma C6885 Dilute stock in PBS to a final concentration of 100mg/ml. Store at -20°C in 100µl aliquots.
Hyaluronidase Sigma H3631 Dilute stock in PBS to a final concentration of 20,000U/ml. Store at -20°C in 100µl aliquots.
100µm cell strainer for 50ml centifuge tube Scientific Lab Supplies (SLS) 352360 Other commercially available cell strainers (e.g. Greiner bio-one) can also be used.
DMEM low glucose Biosera LM-D1102/500 Warm in 37 °C water bath before use.
Penicillin/Streptomycin mix Sigma P4333 Store at -20°C in 1ml aliquots.
25cm2 tissue culture flask SLS 353109
75cm2 tissue culture flask SLS 353136
Bone marrow mesenchymal stem cells vial Lonza PT-2501 Store in liquid nitrogen upon arrival. Cells are at passage 2 upon arrival but are designated passage 0. Exapand to passage 3 and store in liquid nitrogen for later use.
Mesenchymal stem cell growth medium (MSCGM) Lonza PT-3001 Warm in 37 °C water bath before use. For Cell Tracker Green staining use medium without FCS.
EDTA (0.02%) solution Sigma E8008 Store at 4°C. Warm in 37°C water bath before use.
Trypsin solution Sigma T4424 Store at -20°C in 2ml aliquots. Thaw at room temperature and use immediately.
Cryovials Greiner bio-one 2019-02 Keep on ice before adding before adding cell suspension.
Mr. Frosty Freezing Container Nalgene 5100-0001 Store at room temperature. When adding cryovials with cells store at -80°C for 24h before transfrring cells to liquid nitrogen.
Cell tracker green dye Life technologies C2925 Store in 5µl aliquots at -20°C. Dilute in 5ml prewarmed (at 37°C) MSCGM.
Cell counting chambers Nexcelom SD-100 Alternatively a haemocytometer can be used.
Cellometer auto T4 cell counter Nexcelom Auto T4-203-0238
Tumor necrosis factor α (TNFα) R&D Systems 210-TA-100 Dilute stock in PBS to a final concentration of 100,000U/ml. Store at -80°C in 10µl aliquots.
6-well, 0.4µm PET Transwell filters SLS 353090
K2-EDTA in 10ml tubes Sarstedt Store at room temperature.
Histopaque 1119 Sigma 11191 Store at 4°C. Warm to room temperature before use.
Histopaque 1077 Sigma 10771 Store at 4°C. Warm to room temperature before use.
10ml round bottomed tube Appleton Woods SC211 142 AS
7.5% BSA Fraction V solution Life technologies 15260-037 Store at 4°C.
20ml Plastipak syringes BD falcon 300613
5ml Plastipak syringes BD falcon 302187
2ml Plastipak syringes BD falcon 300185
3M hypo-allergenic surgical tape 9m x 2.5cm Micropore 1530-1 Use to secure the syringe tap onto the wall of the perspex chamber.
Silicon rubber tubing, internal diameter/external diameter (ID/OD) of 1/3mm (thin tubing) Fisher Scientific FB68854 Cut silicon tubing to the appropriate size. All tubing leading directly to the electronic microvalve must be thin.
Silicon rubber tubing ID/OD of 2/4mm (thick tubing) Fisher Scientific FB68855
Portex Blue Line Manometer tubing Smiths 200/495/200 Tubing leading to the syringe pump.
3-way stopcock BOC Ohmeda AB
Glass 50ml syringe for pump Popper Micromate 550962 Must be primed prior to use by removing any air bubbles.
Glass coverslip Raymond A Lamb 26x76mm coverslips made to order. Lot number 2440980.
Parafilm gasket American National Can Company Cut a 26x76mm piece of parafilm using an aluminium template and cut a 20x4mm slot into it using a scalpel 10a. Gasket thickness is approximately 133µm.
Two perspex parallel plates Wolfson Applied Technology Laboratory Specially designed chamber consisting of parallel plates held together by 8 screws. The lower plate has a viewing slot cut out in the middle and a shallow recess milled to allow space for the coverslip, filter and gasket. The upper perspex plate has an inlet and outlet hole positioned over the flow channel.
Electronic 3-way microvalve with min. dead space Lee Products Ltd. LFYA1226032H Electronically connected to a 12 volt DC power supply.
Syringe pump for infusion/withdrawal (PHD2000) Harvard Apparatus 70-2001 Set the diameter to 29mm and refill (flow) rate.
L-shaped connector Labhut LE876 To attach to the inlet and outlet ports onto the Ibidi microslide channel.
Video camera Qimaging 01-QIC-F-M-12-C Connected to a computer which enables digitall videos to be recorded.
Image-Pro Plus 7.0 Media Cybernetics 41N70000-61592 For data analysis. Manually tag cells displaying the different behaviors. Track cells for analysis of rolling and migration velocities.

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Citer Cet Article
An In Vitro Flow Investigation of the Effect of Stromal Cells on Leukocyte Recruitment. J. Vis. Exp. (Pending Publication), e22016, doi: (2024).

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