Madin-Darby Canine Kidney (MDCK CCL-34) cells (ATCC, USA) were cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, high glucose pyruvate; Gibco, USA). The DMEM was supplemented with 10% foetal bovine serum (Bovogen Biologicals, Australia), 1x GlutaMAX (Gibco, USA), 1x MEM non-essential amino acid solution (Gibco, USA), 0.05% sodium bicarbonate (Gibco, USA), 20 μM HEPES (Gibco) and 100 U/mL penicillin-streptomycin solution (Gibco, USA). DMEM media was used for virus dilution, containing the above constituents excluding only serum, hereafter known as maintenance media. Maintenance media was also used for virus infection protocols, although media was supplemented with 8 µg/mL TPCK-treated trypsin (SAFC Biosciences, USA), hereafter known as infection media.
Mdck ccl 34 cells
Manufactured by American Type Culture Collection
Sourced in United States
MDCK CCL-34 cells are a well-established cell line derived from Madin-Darby canine kidney. They are commonly used in research applications to study various cellular processes and functions.
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Lab products found in correlation
Sodium bicarbonate, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Mem non essential amino acid solution, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Foetal bovine serum (fbs), by Bovogen (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Acetylated trypsin, by Merck Group (1 mentions)
Gentamicin sulfate solution, by Merck Group (1 mentions)
Heat inactivated fetal bovine serum, by Biosera (1 mentions)
6 protocols using mdck ccl 34 cells
1
MDCK Cell Culture for Virus Studies
2
MDCK and Calu-3 Cell Culture Protocols
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Madin–Darby Canine Kidney (MDCK CCL-34) cells (ATCC, Manassas, VA, USA) were cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, high glucose pyruvate; Gibco, Waltham, MA, USA). DMEM was supplemented with 10% foetal bovine serum (FBS, Bovogen Biologicals, Australia), 1× GlutaMAX (Gibco, USA), 1× MEM non-essential amino acid solution (Gibco, USA), 0.05% sodium bicarbonate (Gibco, USA), 20 μM HEPES (Gibco, USA), and 100 U/mL penicillin-streptomycin solution (Gibco, USA). Maintenance medium (DMEM medium containing the above constituents excluding only serum) was used for virus dilutions. Maintenance medium supplemented with 2 µg/mL TPCK-treated trypsin (Infection medium; SAFC Biosciences, Lenexa, KS, USA) was used for virus infection protocols [3 (link)].
Calu-3 (HTB-55) cells (ATCC, USA) were cultured at 37 °C and 5% CO2 in Eagle’s Minimum Essential Medium (EMEM; Gibco, USA), supplemented with 10% FBS, 50 U/mL penicillin-streptomycin solution, 1× MEM non-essential amino acid solution, and 1 mM sodium pyruvate (Gibco, USA). During infection, Calu-3 medium was supplemented as above, although modified to include 0.3% FBS and 2 µg/mL TPCK-treated trypsin.
Calu-3 (HTB-55) cells (ATCC, USA) were cultured at 37 °C and 5% CO2 in Eagle’s Minimum Essential Medium (EMEM; Gibco, USA), supplemented with 10% FBS, 50 U/mL penicillin-streptomycin solution, 1× MEM non-essential amino acid solution, and 1 mM sodium pyruvate (Gibco, USA). During infection, Calu-3 medium was supplemented as above, although modified to include 0.3% FBS and 2 µg/mL TPCK-treated trypsin.
3
Influenza Virus Propagation in Cell Lines
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Human embryonic kidney (293T) cells and Madin-Darby canine kidney (MDCK) CCL-34 cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco’s Modified Eagle Medium (GIBCO/BRL, Grand Island, NY; catalog number 11965-092) supplemented with 5% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA; catalog number S12450H) and penicillin-streptomycin (Invitrogen, Carlsbad, CA; catalog number 15140122) at 37 °C with 5% CO2. The HA gene of CA/04 was cloned into the vector pHW2000 and used as a template to construct the mutant library. The viruses generated by reverse genetics were propagated in MDCK cells and cultured at 37 °C with 5% CO2 in Opti-MEM medium (GIBCO/BRL, Grand Island, NY; catalog number 11058-021) supplemented with 1 μg/ml of TPCK (N-tosyl-L-phenylalanine chloromethyl ketone)-Trypsin (Sigma-Aldrich, St. Louis, MO; catalog number T1426) and penicillin-streptomycin (Invitrogen, Carlsbad, CA; catalog number 15140122). The virus titers were determined by TCID50 in MDCK cells.
4
Influenza Virus Propagation Protocol
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We used purified human AGP (Sigma–Aldrich; catalog no.: G9885) and the A/Philippines/2/1982 (Phil82) strain of IAV as glycoprotein standards. Phil82 was expressed in chicken eggs and generously provided to us by Dr Kevan Hartshorn at Boston University School of Medicine. The A/Switzerland/9715293/2013 (H3N2) (SWZ13) viral samples, both WT and 5B8 mutant, were propagated in parallel in specific pathogen-free embryonated chicken eggs and MDCK CCL-34 cells (American Type Culture Collection). The protocol for generation of the influenza virus has been described elsewhere (12 (link)). The mutant HA sequence, herein referred to as strain 5B8, had three amino acid substitutions, Q132H, Y219S, and D225N, none of which disrupted any N-glycosylation sequons.
5
MDCK Cell Cultivation and Sialic Acid Variants
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The MDCK cells (CCL-34) were obtained from American Type Culture Collection (ATCC). The wild type MDCK NBL-2 cells (MDCK-wt) and the MDCK cells expressing Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) (MDCK-Gc) were adapted from another study (17 (link)). Limited Neu5Gc (<1%) was detected in MDCK-wt whereas approximately 40% of their total Sia in MDCK-Gc as Neu5Gc (17 (link)). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA, USA) at 37°C under 5% CO2.
6
Influenza A virus propagation in MDCK cells
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MDCK cells (CCL-34) were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in minimum essential medium (Sigma-Aldrich Co. LLC) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (Biosera, Kansas, MO, USA) and 50 μg/mL gentamicin sulfate solution (Fujifilm Wako Pure Chemical Corporation). Influenza A virus (IAV) strain A/Puerto Rico/8/1934 (H1N1; VR-1469) was obtained from the American Type Culture Collection. IAV was propagated at 37 °C using MDCK cells in serum-free medium (SFM; Thermo Fisher Scientific Japan K.K., Kanagawa, Japan) supplemented with acetylated trypsin (2 μg/mL) (Sigma-Aldrich Co. LLC) and gentamicin sulfate solution (50 μg/mL). The virus pellets were obtained according to some previous reports with minor modifications [11 (link),12 (link),13 (link)]. Briefly, the culture medium containing the propagated virus was centrifuged at 300× g for 5 min, the supernatant was then centrifuged at 13,000× g for 2 h, and the supernatant after centrifugation was discarded; the pellet was then re-suspended, and the solution was collected as a condensed viral solution.
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