The HT-29 cell lines used in this work were obtained from the American Type Culture Collection (Rockville, MD) and were cultured at 37°C under 5% CO2 in RPMI 1640 medium supplemented with antibiotics (100 U/ml of penicillin and 100μg/ml of streptomycin) and 10% heat-inactivated fetal bovine serum. After the cells had grown to confluence, they were replated onto 12-mm round coverslips (Warner Instruments Inc., Hamden, CT) and incubated for at least 24 h before use for cytosolic Ca2+ concentration([Ca2+]cyt).
12 mm round coverslips
Manufactured by Warner Instruments
Sourced in United States
12-mm round coverslips are a type of laboratory equipment used to cover and protect samples during microscopy. They are circular glass or plastic discs with a diameter of 12 millimeters, designed to be placed over specimens on microscope slides.
Automatically generated - may contain errors
Lab products found in correlation
Ht29 cell lines, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Iec 6, by American Type Culture Collection (1 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (1 mentions)
75 cm2 flask, by Corning (1 mentions)
Prolong gold with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
4 protocols using 12 mm round coverslips
1
HT-29 Cell Culture Protocol
2
Rat Small Intestinal Cell Culture Protocol
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IEC-6 (ATCC Cat# CRL-1592, RRID: CVCL_0343) rat small intestinal cells were purchased from ATCC and were maintained in DMEM-HIGH GLUCOSE medium (Gibco), containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco) in a humidified 5% CO2 atmosphere at 37 °C. After the cells had grown to confluence, they were replated onto 12-mm round coverslips (Warner Instruments Inc) and incubated for at least 24 h before use for intracellular Ca2+ measurement or immunostaining.
3
Canine Duodenal Epithelial Cell Culture
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SCBN is a non-transformed duodenal epithelial crypt cell line of canine origin. As described previously (Pang et al., 1996 (link); Buresi et al., 2001 (link)), cells in flasks were fed with fresh DMEM supplemented with 10% fetal bovine serum, l-glutamine, and streptomycin every 2–3 days (Buret and Lin, 2008 (link)). Cells of passages 23–33 were grown to confluence (∼5 days) in 75-cm2 flasks (Corning, USA), then replated onto 12-mm round coverslips (Warner Instruments Inc., Hamden, USA) and incubated for at least 24 h before use in pHi measurements.
4
In vivo assembly of sfGFP in J774A.1 macrophages
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To observe in vivo assembly of sfGFP in J774A.1, GFPopt-expressing J774A.1 cells were plated in 12-well plates containing 1.5-thickness 12-mm round coverslips (Warner Instruments, Hamden, CT) at a density of 1 × 105 cells per well. After 24 h, J774A.1 cells were infected with L. pneumophila expressing GFP11, GFP11::pRalF, or NLuc::GFP11 at an MOI of 1. Plates were immediately centrifuged at 930 × g for 10 min and then incubated at 37°C with 5% CO2. At 24 h postinfection, coverslips were fixed with 4% formaldehyde in PBS for 30 min at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 15 min, mounted in ProLong Gold with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA), and visualized with an LSM 880 confocal microscope (Carl Zeiss Microscopy, White Plains, NY) using enhanced GFP and DAPI settings for image collection. Image analysis was performed using Zen 2.1 software.
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