Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Tyrodes Salt Solution

NaClxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 8.00 g

KClxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 0.20 g

CaCl₂xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 0.20 g

MgCl₂ · H₂Oxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 0.05 g

NaH₂PO₄ · H₂Oxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.00 g

NaHCO₃ · H₂Oxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.00 g

Glucosexa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.00 g

Glass distilled H₂O toxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.00 L

Add ingredients in the sequence listed.xa0 Filter-sterilize.

1.xa0xa0 Harvest the parasites according to the protocol for maintenance in vivo.

2.xa0xa0 Spin the cell suspension at approximately 50 x g for 3 min, to remove any cellular debris.

3.xa0xa0 Transfer the supernatant to a new 15 ml plastic centrifuge tube.xa0 Centrifuge at 1300 x g for 10 min.

4.xa0xa0 Pool the cell pellets and adjust the concentration to 2.0 - 4.0 x 10⁷ cells/ml with a fresh solution of Tyrodes Salt Solution.

xa0xa0xa0xa0xa0 *If the concentration is too low centrifuge at 1300 x g for 10 min and resuspend in the volume of Tyrodes Salt Solution required to yield the desired concentration.

5.xa0xa0 Mix the cell preparation and 10% (v/v) DMSO in equal portions.xa0 The final concentration will be 1.0 - 2.0 x 10⁷ cells/ml and 5% DMSO.xa0 The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.

6.xa0xa0 Dispense in 0.5 ml aliquots to 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

7.xa0xa0 Place the vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If the freezing unit can compensate for the heat of fusion, maintain rate atxa0xa0xa0xa0xa0xa0xa0 -1°C/min through the heat of fusion.xa0 At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately

-1°C/min.) xa0

8.xa0xa0 Store in either the vapor or liquid phase of a nitrogen refrigerator.

9.xa0xa0 To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.xa0 Do not agitate the ampule.xa0 Do not leave ampule in water bath after thawed.

10. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected mouse.xa0 Follow the protocol for maintenance in vivo.