Optimization of the method for analyzing endocytosis of fluorescently tagged molecules: Impact of incubation in the cell culture medium and cell surface wash with glycine-hydrochloric acid buffer

To obtain the therapeutic effect of biological medicines, such as proteins and nucleic acids, these medicines must achieve their intracellular target, such as the cytoplasm, and pass through biological membrane barriers. Endocytosis is an attractive route for the intracellular delivery of such drugs, and various endocytosis inhibitors have been used as tools to study the involvement of endocytosis in the cell internalization of delivery carriers. However, the specificity of these inhibitors has been insufficiently studied, and our preliminary tests could not detect the expected effect of the well-known endocytosis inhibitors.
Therefore, the present study aimed to optimize the experimental conditions to precisely analyze cellular internalization via endocytosis. We first found that incubation of model molecules, such as transferrin (Tf) and cholera toxin subunit B (CTB), in cell culture medium (DMEM) could efficiently induce their internalization to HeLa cells compared to that in transport buffer (HBSS). Moreover, we clarified that cell surface wash with glycine-hydrochloric acid buffer before confocal microscopy and flow cytometry strengthened the intracellular fluorescence of Tf, CTB, and dextran tagged with fluorescent probes possibly via the neutralization of endosomal pH. Even under the optimized condition, however, the specificity of endocytosis inhibitors was disputable. The present study suggested the importance of the optimization of the study design with endocytosis inhibitors in analyzing cellular internalization.

The adequate amount of sodium chloride in Protein A wash buffer for effective host cell protein clearance.

Post-load column wash in Protein A chromatography can effectively improve host cell protein (HCP) clearance. A commonly used wash additive for this purpose is sodium chloride. However, the adequate amount of sodium chloride required for effective HCP clearance is less consistent in literature. In this study we investigated the impact of different amounts of sodium chloride on HCP clearance with five monoclonal antibodies (mAbs).
For each mAb, elution pool HCP levels from runs under different wash conditions are compared. For all five mAbs, the data suggested that 250 mM would be an adequate amount for the salt to largely achieve its HCP reducing effect. The same conclusion is also reached for calcium chloride, a less commonly used but equally effective Protein A wash additive for HCP clearance.

Inactivation of viruses using novel protein A wash buffers.

  • Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc-fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc-fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3.
  • The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied.
  • The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation >> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities.

Maternal plasma or human serum albumin in wash buffer enhances enrichment and ex vivo expansion of human umbilical cord blood CD34+ cells.

Umbilical cord blood is a valuable source of haemopoietic stem/progenitor cells (HSC) for transplantation. This study explored the effect of maternal plasma/human serum albumin (HSA) in the purification and culture conditions of CD34+ cells derived from human umbilical cord blood. During CD34+ cell enrichment, including maternal plasma or HSA instead of fetal bovine serum (FBS) in the wash buffer, significantly increased the purity and the fold expansion of CD34+ cells. The increase in fold expansion of CD34+ cells was independent of CD34+ cell purity before expansion. With FBS, the mean fold expansion of CD34+ cells and total nucleated cells on day 7 was 9.7 +/- 5.5 and 39.7 +/- 13.7 respectively.
The use of maternal plasma increased the mean fold expansion of CD34+ cells and total nucleated cells on day 7 to 28.2 +/- 6.7 and 71.5 +/- 15.4 respectively. When HSA was added to wash buffer, the mean fold expansion of CD34+ cells and total nucleated cells were 30.4 +/- 10.5 and 83.5 +/- 24.8 respectively. No statistical significance was found between using HSA and maternal plasma on total cell and CD34+ cell expansion. We propose that HSA in maternal plasma was responsible for the positive effect on CD34+ cell enrichment and expansion.

Enhanced removal of detergent and recovery of enzymatic activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis: use of casein in gel wash buffer.

The inclusion of 1% casein or bovine serum albumin in buffer used to reactivate enzymes subjected to sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis resulted in accelerated removal of SDS and restoration of nuclease and beta-galactosidase enzyme activities. Nuclease and beta-galactosidase activities which are absent from gels after longer wash procedures are detectable with this technique. Enzyme activity in gels prepared with SDS which contained inhibitory contaminants was partially restored by the casein wash procedure. The threshold of detection of two-dimensionally separated deoxyribonuclease I using the casein wash procedure was 1 picogram.

Wash Buffer

1210-200 Biovision each 235.2 EUR

Wash Buffer

abx293002-30ml Abbexa 30 ml 126 EUR

Wash Buffer

abx098952-20ml Abbexa 20 ml 109.2 EUR

Wash Buffer

KF17356 Neuromics 500 ml 214.8 EUR

WASH BUFFER

IB47092 IBI Scientific 25ML 96.49 EUR

Wash Buffer

abx095521-100l Abbexa 100 µl 93.75 EUR

Wash Buffer

abx095521-1ml Abbexa 1 ml Ask for price

Wash Buffer

abx095521-200l Abbexa 200 µl Ask for price

Wash Buffer (25X)

abx092104-30ml Abbexa 30 ml 126 EUR

Wash Buffer (20x)

RM00026 Abclonal 30mL 280 EUR

10X Wash Buffer

DS0230-100ML EWC Diagnostics 1 unit 2.57 EUR

10X Wash Buffer

DS0230-500ML EWC Diagnostics 1 unit 11.68 EUR

Block Wash Buffer

I044 Cygnus Technologies 1000 ml 632.4 EUR

Wash Buffer, pH 7.4

40121142-1 Glycomatrix 250 mL 15.55 EUR

Wash Buffer, pH 7.4

40121142-2 Glycomatrix 500 mL 35.69 EUR

Wash Buffer, pH 7.4

40121142-3 Glycomatrix 1 L 53.99 EUR

Wash Buffer, pH 8.0

42320003-1 Glycomatrix 500 mL 36.41 EUR

Wash Buffer, pH 8.0

42320003-2 Glycomatrix 1 L 72.64 EUR

Permeabilization Wash Buffer

E16FXP009 EnoGene 150 ml 86.67 EUR

ChIP Wash Buffer A

10450060-1 Glycomatrix 100 ml 34.32 EUR

ChIP Wash Buffer A

10450060-2 Glycomatrix 250 ml 54.54 EUR

ChIP Wash Buffer B

10450061-1 Glycomatrix 100 ml 34.32 EUR

ChIP Wash Buffer B

10450061-2 Glycomatrix 250 ml 54.54 EUR

Gel Wash Buffer (BIW)

DS0030-10ML EWC Diagnostics 1 unit 13.07 EUR

TSM Wash Buffer TSMW

40120101-1 Glycomatrix 100 mL 16.68 EUR

TSM Wash Buffer TSMW

40120101-2 Glycomatrix 500 mL 36.33 EUR

Development of a cell wash buffer that minimizes nucleic acid loss from Clostridium perfringens 10543 A.

Autolytic activity and nucleic loss from Clostridium perfringens 10543 A was demonstrated during successive cell washes in hypotonic TES buffer. Autolysis increased nearly sixfold and nucleic acid loss nearly twofold when 10 mM EDTA was added to 0.3 M Tris-sucrose buffer.
Attempts to minimize both autolysis and nucleic acid loss from C. perfringens during routine washing steps were unsuccessful when the effects of sucrose concentration, pH, CaCl2 addition, or wash temperature were examined independently. However, autolytic activity was eliminated and nucleic acid loss reduced to less than 5% when C. perfringens cells were washed at 4 or 25 degrees C in 1.0 M sucrose, 50 mM Tris–HCl, and 25 mM CaCl2 at pH 5.7.

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