Murine RPCs were isolated from the peritoneal lavage fluid of naive mice as previously described (2 (link), 4 (link)). Naive mice (n = 40) were anesthetized using CO2 and sacrificed by cervical dislocation. The abdominal cavity was flushed with 5 mL Ca2+/Mg2+-free DPBS (Gibco, Waltham, MA, USA). The collected peritoneal lavage fluid was centrifuged at 400 g for 10 min at 4°C, and the pelleted RPCs were resuspended in complete RPMI-1640 (Gibco) medium [RPMI-1640 medium containing 0.05 mM 2-β-mercaptoethanol (Sigma-Aldrich), 3 mM L-glutamine (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), 100 U/mL penicillin/streptomycin (Sigma-Aldrich), and 10% fetal calf serum (HyClone)] and incubated at 37°C with 5% CO2 (4 (link)). All cells were freshly isolated prior to use.
Ca2 mg2 free dpbs
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ca2+/Mg2+-free DPBS is a phosphate-buffered saline solution that does not contain calcium or magnesium ions. It is commonly used in cell culture and various laboratory procedures where the absence of these ions is required.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
2 β mercaptoethanol, by Merck Group (1 mentions)
Heparin tube, by BD (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Histopaque, by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Tc20 automated cell counter, by Bio-Rad (1 mentions)
Vacutainer heparin tube, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
4 protocols using ca2 mg2 free dpbs
1
Isolation of Murine Resident Peritoneal Cells
2
Isolation of PBMCs from Pigs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were individually isolated from the whole blood samples (20 mL/donor) collected from FMD antibody-negative pigs (8–9 weeks-old, n = 3/group) in heparin tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), using Histopaque-1077 (Sigma-Adrich) as previously described (4 (link), 25 (link)). The remaining red blood cells were then removed by lysis using ACK lysing buffer (Gibco). Thereafter, PBMCs were resuspended in Ca2+/Mg2+ free DPBS (Gibco) for washing. The purified PBMCs were incubated in complete RPMI 1640 medium as mentioned in section 2.4 RPC Isolation and Culture and incubated at 37°C with 5% CO2 (4 (link)).
3
Isolation and Culture of Porcine PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were isolated from whole blood samples collected from pigs (n = 5–6/group) in heparin tubes, using Histopaque (Sigma-Aldrich). The remaining red blood cells (RBCs) were then removed by lysis using ACK lysing buffer (Gibco). Thereafter, PBMCs were resuspended in Ca2+/Mg2+ free DPBS (Gibco) and stored until use. Isolated PBMCs were incubated in RPMI 1640 (Gibco) containing 10% FBS and 1% antibiotic-antimycotic (Gibco) at 37 °C and in a 5% CO2 atmosphere.
4
Isolation of Porcine PBMCs for Immunological Studies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Porcine PBMCs were isolated from whole blood of FMD antibody-seronegative pigs as donors (8-9 weeks old animals, n = 3/group) according to the method described by Lee et al. and Jo et al.17 ,67 (link). Whole blood (20 mL/donor) was independently collected in BD Vacutainer heparin tubes (BD, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) gradient centrifugation. Residual red blood cells were lysed with ammonium–chloride–potassium (ACK) lysing buffer (Gibco). The PBMCs were suspended in Ca2+/Mg2+-free DPBS (Gibco) and counted using a Bio-Rad TC20 Automated Cell Counter (Bio-Rad). All cells were freshly isolated before use. No cryopreserved cells were used in any experiment. Purified PBMCs were then resuspended in RPMI-1640 (Gibco) medium supplemented with 10% FBS (Gibco), 3 mM L-glutamine (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), and 100 U/mL penicillin–streptomycin (Sigma-Aldrich). Incubations were carried out at 37 °C and 5% CO2.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!