Techniques for Dual Staining of DNA and Intracellular Immunoglobulins in Murine Hybridoma Cells: Applications to Cell-Cycle Analysis of Hyperosmotic Cultures

Abstract

Flow cytometry was used to evaluate the effects of hyperosmotic stress on cell-cycle distribution and cell-associated immunoglobulins for murine hybridoma cells grown in batch culture. Paraformaldehyde/methanol fixation substantially increased the fluorescence signal for intracellular immunoglobulins compared to ethanol fixation. For surface immunoglobulins, similar fluorescence signals were observed regardless of fixation method. Dual staining of immunoglobulins and cellular DNA was employed to determine immunoglobulin pool size as a function of cell-cycle phase. The intracellular immunoglobulin pool sizes increased as the cells progressed through the cell cycle for both control and hyperosmotic cultures. For control cultures, the immunoglobulin pool size increased during the exponential phase of culture, followed by a decrease as the cultures entered stationary phase. In contrast, hyperosmotic cultures showed an initial decrease in immunoglobulin pool size upon the application of osmotic shock, followed by an increase to a level above that of control cultures. This behavior was observed in all phases of the cell cycle. In addition, hyperosmotic cultures exhibited an increase in cell size when compared to control cultures. When normalized for cell size, the intracellular immunoglobulin concentration in hyperosmotic cultures was initially lower than in control cultures and subsequently increased to slightly above the level of control cells. Cells in all phases of the cell cycle behaved in a similar manner. There was no apparent relationship between the intracellular antibody concentration and the rate of antibody secretion.

Using Molecular Markers to Characterize Productivity in Chinese Hamster Ovary Cell Lines

“…After incubation, the cells were washed with cold PBS and resuspended in cold PBS. The HC and LC fluorescence emissions were measured with a 530/30 bandpass (BP) filter [16], [43], [44].…”

Detailed understanding of enhanced specific antibody productivity in NS0 myeloma cells

“…The intracellular heavy chain and light chain concentration was measured by flow cytometry using a protocol established by McNeely et al (2005) and calculated based on methods established by Borth et al (1999) and Strutzenberger et al (1999). Cells samples were centrifuged to remove supernatant and washed with sterile cold PBS.…”

Short‐ and long‐term effects on mAb‐producing CHO cell lines after cryopreservation

“…To perform dual staining of DNA and intracellular mAb, the propidium iodide (PI) fluorescent dye (Thermo Fisher Scientific) for detecting DNA was used in conjunction with the FITC‐conjugated goat anti‐human IgG (H + L) F(ab′) 2 described above. This dual staining was performed based on a previously published method . In brief, intracellular mAb staining was first performed as described above.…”

“…This dual staining was performed based on a previously published method. 17 In brief, intracellular mAb staining was first performed as described above. After the final PBS wash, the cells were resuspended in RNase A solution (0.2 mg/mL; Sigma-Aldrich) and incubated (room temperature, 30 min).…”

“…This observation is not surprising because fluorescence intensity on the histograms is expressed on a log scale; the width of the base for a single peak can extend beyond a 10-fold (i.e., 1 log) range in fluorescence intensity, as observed for intracellular IgG expression in recombinant CHO cells (Figure 9, Supporting Information Figures S1-S5) 10 and hybridoma cells. 14,17 In addition, the double peaks observed in recombinant CHO cells (Figure 9 and Supporting Information Figure S3) typically displayed a peak-to-peak separation in fluorescence intensity of 1-2 log. 10 Attempts to increase productivity of CHO cells by cell cycle control have yielded up to several-fold increase in Q p .…”