Our protocol provides a cocktail medium to prolong the neutrophil lifespan to more than five days without compromising the neutrophil functions. It also offers reliable in vitro dialysis for neutrophils. CLON-G treatment expands the possibilities of neutrophil preservation, transportation, and gene manipulation, which can accelerate research in the neutrophil community.
To begin, prepare the basic medium by adding RPMI 1640 FBS and pen-strep to a 50 milliliter tube. Simultaneously, thaw all the stock solutions of the CLON-G components on ice. Then dilute HSP 70 and 200 micrograms per milliliter of G-CSF in the basic medium.
Transfer 976 microliters of basic medium to a 15 milliliter centrifuge tube. To this, add the CLON-G components to prepare one milliliter of 2x CLON-G medium as described in the manuscript. Next, prepare the neutrophil culture by suspending the isolated mouse neutrophils in the basic medium.
Add CLON-G medium, followed by the basic medium to 24 well non tissue cultured plates. Then add 100 microliters of the prepared cell suspension and gently pipet three to four times. Incubate the culture plate at 37 degrees Celsius with 5%carbon dioxide supply.
Stain the cultured neutrophils with cytospin and analyze them using a light microscope. For flow cytometry analysis, add one milliliter of cell medium to the neutrophil cell culture grown overnight. Mix the cell culture medium by gently pipetting it five to seven times and transfer 100 microliters of this mixture to a flow cytometry tube at the desired time points.
Simultaneously, vortex the counting beads and pipet 10 microliters of bead solution to the same flow cytometry tube containing cell medium. Add 10 microliters of a pre-prepared staining mix to this flow cytometry tube and incubate for five minutes in the refrigerator under dark. Then add 100 microliters of saline to the tube and mix well for even distribution of the counting beads and cells.
Finally, perform the flow cytometry analysis of the cell suspension as described in the text manuscript. For fluorescent images assay, add two milliliters of cell suspension per confocal plate and incubate at 37 degrees Celsius and 5%carbon dioxide. Meanwhile, dissolve one gram of calcium chloride in 45 milliliters of saline and filter it with a 0.2 micrometer filtering unit.
Gently remove the confocal plate at the required time point. Stain the cells with 10 microliters of FITC-Annexin-V, PI and calcium chloride for five minutes at room temperature. Then using a confocal fluorescence microscope, capture the images using the desired channels.
In the present study, the morphology of neutrophils was not affected in the CLON-G medium even after five days. Moreover, the fax phenotypes demonstrated intact cells after the treatment. The flow cytometry analysis showed a positive population of untreated neutrophils in Annexin-V and the population loss could be due to ethylene diamine tetraacetic acid in the cell medium.
However, the viability of the CLON-G treated neutrophils at 24 hours was approximately more than 90%The fluorescent image assays also confirmed the cell viability of CLON-G treated neutrophils. The neutrophils cultured in the basic medium for 24 hours showed puffed cells and the loss of these cells might have resulted from the shaking or pipetting of the confocal plate. CLON-G compound concentrations and effects can significantly impact neutrophil tests.
If at all possible, avoid modulating them. Neutrophil investigations are laborious, expensive, and extremely limited in clinical application due to their short life span. This method partially overcomes these obstacles.
