Serum heat inactivation

Sheep serum. Heat-inactivate by incubating in a water bath at 55°C for 30 min. Store in appropriate amount of aliquots at -20°C. Do not freeze-thaw repeatedly. 

NBCS 20% (v/v) newborn calf serum (heat-inactivated) in TBS. 

Cultures. Swiss 3T3 and A431 cells were grown in a gassed (5.5% COJ, humidified incubator in Dulbecco s Modified Eagle Media (DME) supplemented with 10% heat inactivated fetal calf serum (FCS). 

Culture of Pheochromoqtoma Cells (PC12 Cells). PC12 cells, kindly supplied by Dr. H. Hatanaka of our institute, were maintained in Dulbecco s modified Eagle s medium containing 5% heat-inactivated horse serum. 

Figure 9.5 Healthy volunteer monocytes after erythrophagocytosis. Rabbit erythrocytes were incubated with heat-inactivated mouse antirabbit erythrocyte serum. Human peripheral blood monocytes (MN) were obtained after Ficoll-Paque isolation and monocyte clumping with subsequent separation from lymphocytes, yielding a 95 % pure MN population. Non-ingested erythrocytes were removed by hypotonic lysis.

Add 5 ml Antibiotic-Antimycotic (lOOx, cat. 15240-062) and 50 ml heat-inactivated horse serum (cat. 16050-122) to make complete MEM (used for neuron plating and glial culture). For neuron plating, the complete MEM could be kept for a couple of months at 4°. However, for glial culture, a freshly thawed serum aliquot should be used for each culture. Obvious growth retardation in glial culture is observed when using 1-month-old complete MEM stored at 4°. 

Growth medium RPMI-1640 with L-glutamine (Irvine Scientific Co., Irvine, CA), supplemented with 10% heat-inactivated fetal bovine serum, lOIU/mL penicillin, lOOpg/mL streptomycin (PEST), 50pM2-mercaptoethanol, 292 igl mL L-glutamine, and lOmM HEPES buffer (all from Sigma-Aldrich). 

In general, serum should be heat-inactivated by heating at 56°C for 15 min to inactivate complement components prior to ammonium sulfate fractionation. Ascitic fluid should first be filtered through a cushion of glass wool. 

DMEM high glucose, trypsin-EDTA solution, fetal bovine serum (FBS), heat inactivated (all from Invitrogen). 

Block the embryos with 10% heat inactivated sheep serum in TEST for 1 h (reeNote 16). Prepare the blocking solution in the screw-cap tubes and transfer the embryos. 

Replace the blocking solution with the antibody solution (1% heat inactivated sheep serum in TEST containing 1 2,000 dilution of anti-DIG antibody). It is easy to conduct this process by transferring the embryos with pipette after pouring them out into the petri-dish. 

Aliquots (420 pi) of the emulsion will be preincubated for 90 min at 37°C with 80 pi of heat-inactivated pooled human serum as a source of apoCII [40]. 

Fetal calf serum (FCS) inactivated by heating for 30 min at 5-6°C and tested for ability to support growth of hybridomas (see Note 5). 

Heat-inactivated serum from the second antibody species (not for use with the protein A-gold technique)... 

Wipe excess liquid from the area around the sections, and apply sufficient 5% heat-inactivated normal serum from the second antibody species to cover each entire section (15 min). If protein A-gold probes are used, flood the sections with BSA-PBS rather than normal serum. It is important to keep the sections covered and in a humid environment throughout all incubations, preferably using a purpose-made humidity chamber. Depending on the size of the section, about 50-200 uL of reagent is sufficient to cover each preparation... 

They are incubated in the primary antibody (PC10, diluted 1 200 in PBS containing 0.2% BSA for PCNA) for 1-2 hr at room temperature. The sections are washed five times for 5 min each with PBS and then incubated in the enzyme-conjugated secondary antibody in PBS containing 1% heat-inactivated normal rat serum for 1 hr. Horseradish peroxidase... 

Figure 7. Effects of various sera on ODC activity in KB cells. To log-phase KB cells, fresh media containing (a) no serum, (b) 10% calf serum, (c) 10% heat-inactivated calf serum, or (d) 10% dialyzed calf serum was added ft= 0). ODC activity then measured every 2 hr under each condition listed.

Human blood samples can rather easy be converted to human serum with the use of BD Vacutainer SST centrifugation tubes (Becton Dickinson Diagnostics, Franklin Lakes, NJ). If serum purification is considered over custom orders, the reader should consider pooling serum from several individuals. The serum should also be heat inactivated at 56°C for 30 min. Inactivation may cause precipitation thus sterile filtration of the serum is recommended. 

HEK 293 cells are grown in Dulbecco s MEM / Nut Mix F-12 supplemented with 2 mM L-glutamine, 100 U/mL penicillin-streptomycin and 10% heat-inactivated fetal bovine serum (FBS complete medium). 

Chemotaxis medium RPMI 1640 containing 25 mM HEPES and either 0.5% heat-inactivated fetal calf serum (for lymphocyte chemotaxis) or 1% bovine serum albumin (BS A Fraction V, Sigma A9306) (for monocyte and granulocyte chemotaxis). 

The ensuing sheep serum is heat-inactivated by incubation in a waterbath at 56°C for 30 min, absorbed with rat red blood cells and serum proteins, and filter-sterilized. For storage, the serum is lyophilized and frozen at -70°C. As reported elsewhere (6), NTS has moderate reactivity to type IV collagen and laminin, and substantial reactivity to glomerular cell membrane proteins, particularly pi integrin and its accompanying a chain. 

Improved Minimum Essential Medium (IMEM Biofluids, Inc., Rockville, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum, 100 p,g/mL streptomycin, lOOU/mL penicillin, and 2mM glutamine. 

The method contains three stages including first establishing a cell line followed by test chemical exposure and finally evaluated for expression of cell surface markers. To establish a cell line, human leukocyte preparations are attained from a plasma distributor. The leukocyte preparations as described by Ryan et al. (2004), are diluted with an equal part of complete medium (RPMI 1640 containing 1 x L-glutamine, 1 x penicillin-streptomycin-neomycin antibiotic mixture), 30 p,2-mercaptoethanol and 10 % heat inactivated fetal bovine serum. The diluted preparation is layered onto a Ficoll-Paque gradient to... 

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