Dear Salih!

Please You look at the following protocol:

https://www.nature.com/articles/s41598-020-63344-6

Analysis of C. albicans persistence in co-culture with human leucocytes

Blood samples were obtained from sixteen healthy volunteers by venipuncture. Three independent blood samples were obtained from each subject to evaluate the intra and inter-individual variation of immune composition as previously described32. Briefly, peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophils (PMN) were subsequently separated by dextran sedimentation (Dextran 500, 8% (w/v), density 1.113 ± 0.001 g/ml) followed by gradient centrifugation (dextran:blood ratio of 1:1). PBMC and PMN cell suspensions were washed twice in RPMI 1640, 8% human serum (HS) by centrifugation. Total leucocytes were adjusted to a final concentration of 2.5 × 106 cells/ml and cultured in RPMI 1640, 8% HS in 24-well tissue culture plates. Freshly cells were immediately infected by three C. albicans clinical isolates Caal93, Caal121 and Caal123 from the Mycobank of the Parasitology and Medical Mycology Department, Nantes, France at a monocyte:blastoconidia ratio of 2000:1. Co-cultures were incubated at 37 °C, 5% CO2. Uninfected leucocytes were used as control of cellular reaction. The fungal burden within leucocyte cocultures was followed by colony-forming units calculation and human immune cell composition was analyzed over time by flow cytometry on days 0, 2, 4 and 6 post-infection. The total number and viability of cells at each time point was assessed by cell counting in the presence of 0.5% eosin.

To validate the IL-17 production by human neutrophils, these cells were sorted from peripheral blood cell samples from three healthy subjects by gradient sedimentation according to protocols30. The higher band of PBMC was separated from a lower one of neutrophils. Fresh separate neutrophil and PBMC fractions were simulated with C. albicans strains for two days.

For determination of growth rates, C. albicans clinical isolates were grown overnight at 30 °C in liquid YPD medium. 103 yeasts/ml were suspended in fresh RPMI 1640, 8% HS medium and incubated at 37 °C, 5% CO2 over 12 hours. Growth rates were determined by measuring OD600 every two hours. The doubling time was calculated using the OD600 at 12 hours (T1) compared to h = 0 (T2) as following: ln 2 × 60/((ln OD600 of T1 − ln OD600 of T0)/12).

Article Interaction of Candida albicans with Adherent Human Peripher...

Gamma irradiation of PBMCs.

Gamma irradiation causes DNA damage to PBMCs, yet cells retain intact membrane proteins (3). Irradiated cells are no longer proliferative and do not produce de novo protein but otherwise remain functional. Because the cell membrane and mitochondria remain intact, the cells retain the Mitotracker dye. These gamma-irradiated PBMCs are commonly used as feeder cells in propagating growth of immune cells in vitro (16, 36). To study whether surface molecules on PBMCs affect the growth of C. albicans as biofilms, we irradiated PBMCs with 3,000 rads and then incubated them with C. albicans at the initiation of biofilm formation (adhesion phase). These biofilms were then allowed to grow to the mature phase as described above.

Thank you Dr. Alexandr chernov to answer my question