this presentation is a summarize form of an article to easily deliver the methodology followed in an article. dendritic cells are our very effective cells these are not only to present antigen but also to kill them or process them by their phagocytic activities (phagocytosis) .

1. NEHA HANIF

2. What are dendritic cells?
Dendritic cells (DCs) are immune cells that form part of the mammalian immune
system. Their main function is to process antigen material and present it on the
surface to other cells of the immune system, thus functioning as antigen-
presenting cells.

3. C.neoformans
•Cryptococcus neoformans is an opportunistic fungal pathogen that causes disease
predominantly in immunocompromised patients, particularly individuals with
AIDS, transplant recipients, and those with lymphoid and hematological
malignancies
•Protective immunity to C. neoformans is dependent on an adaptive Th1-type
immune response .

4. How dendritic Cells Kill The Pathogen?

5.  tissue culture media RPMI
 Media for human dendritic cell generation consist of RPMI-1640
supplemented with 10% heat-inactivated fetal bovine serum, 2mM
L-glutamine,100 U penicillin/ml,100 µg of streptomycin/ml and
50mM,2-mercaptoethanol (complete medium).
 plastic ware
 OREGON green labelled 3C2 antibodies

6.  . Yeast cells were grown for 18 hours at 30°C
with shaking in YPD broth.
 Cells were centrifuged and washed three
times with sterile phosphate-buffered saline
(PBS). Viable yeasts were quantified using
trypan blue dye exclusion in a
hemacytometer.

7.  peripheral blood was obtained from healthy volunteers by veni-
puncture
 The blood was anti-coagulated with heparin
 The tubes were centrifuged at 1,000 × g for 10 min without the
brake.
 After that plasma was collected and stored at −20°C until use.

9. 1ug green oregon
stained 3c2 antibodies
Dendritic cells
Cryptococcus
neoformans
PHAGOCYTOSIS OF CRYPTOCOCCUS BY DENDRITIC
CELLS
Incubate 25 ◦C for 10 to 60
minutes
4 TUBES of 1.7ml RPMI(Roswell Park Memorial Institute)
(labelled as 10 min,20min,30min or 60 min)

10. Confocal microscopy
PARA-FORMALDEHYDE
2%
Leave at
room temp
for 10 min
Saponin 0.1%
Leave at
room temp
for 10 min
INTRACELLULAR STAINING
EEA-1 ANTIBodies
EACH TUBE

11. Extraction of lysosome
lysosomal extraction
buffer 2.7 mlDendritic cells Homogenizer (20-25
time)
RPMI
CENTRIFUGE :1000
rpm for 10 min
Centrifuge at 20000 rpm for 20 min Collect supernatant

12. 1ml
Sonicate-20sec
Incubate at room
temp for 3 days
PELLET CONTAIN
LYSOSOME
LYSOSOME EXTRACTED
TAKE C.NEOFORMANS CULTURE
INOCULATE IN PBS
LYSOSOMAL
EXTRACTS

13. DILUTE IN PBS ,PLATED ON SDA AND CALCULATE CFU

14. RESULTS

15.  the intracellular staining confirmed the entry
of C. neoformans organisms into endosomes
and lysosomes.
 The results OF CONFOCAL MICROSCOPY
showed that the majority of C.
neoformans organisms appeared to enter the
lysosomal compartment of HDCs within 20
min following uptake.

16. We determined whether lysosomal components
from DCs were capable of killing C.
neoformans. We found that crude lysosomal
extracts from DCs had dose-dependent anti
cryptococcal activity, with nearly complete
killing observed when the extracts were diluted
50%.