Biomedical Engineering Reference
In-Depth Information
2
Materials
All solutions are prepared using double distilled water and may be
kept at room temperature (RT) unless otherwise indicated. Please
read all necessary material safety data sheets (MSDS) and take
appropriate safety precautions before beginning this protocol.
Follow all guidelines for responsible waste disposal.
2.1 Materials
Required for
Preparation of SLO
Stock Solution
1. Phosphate buffered saline solution (PBS, 140 mM NaCl,
2.7 mM KCL, 1.4 mM KH
2
PO
4
, 0.8 mM Na
2
HPO
4
, pH 7.4).
2. Streptolysin O (SLO, Sigma S-5265, or
see
Note 1
). Dissolve
the contents of a vial of SLO in 2.25 ml of PBS. Store 90
ʼ
l
aliquots at −80 °C (
see
Note 2
).
2.2 Materials
Required for Assaying
Hemolytic Units (HU)
1. PBS as per Subheading
2.1
.
2. Human erythrocyte concentrate (Buffy coat removed, usually
available from local blood bank).
3. Two aliquots of SLO prepared as in Subheading
2.1
above.
4. 1 M Dithiothreitol (DTT). Freeze aliquots and thaw fresh for
each assay.
5. Microcentrifuge.
6. Plate reader capable of measuring absorbance at 412 nm.
7. Microtiter plate suitable for plate reader above.
2.3 Materials
Required for SLO Lysis
and Sample Preparation
1. SLO prepared as in Subheading
2.1
above.
2. 1 M DTT as above.
3. PBS as per Subheading
2.1
4. 2 × 10
8
(20
l packed volume) early to mid trophozoite-stage
(approx. 24 h post invasion) infected erythrocytes purifi ed to
at least 50 % parasitemia (
see
Note 3
). Determine parasitemia
by counting Giemsa-stained slides.
5. Microcentrifuge.
6. Reducing sample buffer (4×): 200 mM Tris/HCl pH 6.8,
10 mM EDTA, 40 % glycerol, 8 % SDS, 0.04 % bromophenol
blue, 200 mM dithiothreitol. Store aliquots at −20 °C for up
to a month.
ʼ
2.4 Materials
Required for Protease
Protection Assay
of SLO Permeabilized
Infected Erythrocytes
1. PBS as per Subheading
2.1
.
2. Proteinase K (10 mg/ml in PBS, make fresh every time, keep
on ice until required).
3. 10 % Triton-X-100 in PBS.
4. 100 mM Phenylmethanesulfonyl fl uoride (PMSF)/
Isopropanol. Add 0.174 g PMSF to 10 ml of 100 % Isopropanol
and allow to dissolve. Keep frozen at −20 °C. Allow to come to
room temperature before use.
